Methods of treating neoplasia with combinations of target cell-specific adenovirus and chemotherapy

ABSTRACT

The invention provides methods of treating neoplasia using combinations of target cell-specific replication competent adenoviral vectors and chemotherapy, radiation therapy or combinations thereof. The adenoviral vectors are target cell-specific for the particular type of neoplasia for which treatment is necessary and the combination with the chemotherapy and/or radiation leads to synergistic treatment over existing adenoviral therapy or traditional chemotherapy and radiation therapy.

TECHNICAL FIELD

This invention relates to cell transfection, and in particular methodsof using adenoviral vectors for the suppression of tumor growth inconjunction with chemotherapy, radiation therapy or combinationsthereof.

BACKGROUND ART

Neoplasia, also known as cancer, is the second most common cause ofdeath in the United States. While the survival rates for individualswith cancer have increased considerably in the last few decades,survival of the disease is far from assured. Cancer is a catch-all termfor over 100 different diseases, each of which are each fundamentallycharacterized by the unchecked proliferation of cells. Individual cancercells are also able to break off from the main tumor, or metastasize,creating additional tumors in other regions of the body.

Due to the mortality rate and incidence of neoplasia in the generalpopulation, research into potential cures has been high on the nationalagenda for decades. This research has led to the development a number oftreatments, both systemic and regional (local). Regional treatmentsinclude radiation therapy, some types of chemotherapy and surgery.Chemotherapy has most often been used in systemic treatment. Each ofthese treatment regimes has significant disadvantages and limitations.Chemotherapy and radiation treatments will be discussed below.

Chemotherapy

Chemotherapy refers to the use of chemical compounds or drugs in thetreatment of disease, though the term chemotherapy is most oftenassociated with the treatment of cancer. Cancer chemotherapeutic agentsare also commonly referred to as antineoplastic agents. There are anumber of classes of chemotherapeutic compounds, encompassing nearly 100individual drugs, as well as numerous drug combination therapies,methods of delivery and schedules of treatment. Each of thesechemotherapeutic agents may be classified according to several criteria,such as class of compound and disease state treated. Certain agents havebeen developed to take advantage of the rapid division of cancer cellsand target specific phases in the cell cycle, providing another methodof classification. Agents can also be grouped according to the type andseverity of their side effects or method of delivery. However, the mostcommon classification of chemotherapeutic agents is by class ofcompound, which broadly encompasses the mechanism of action of thesecompounds.

Depending on the reference source consulted, there are slightdifferences in the classification of antineoplastics. The classes ofcompounds are described in the Physician's Desk Reference as follows:alkaloids; alkylating agents; anti-tumor antibiotics; antimetabolites;hormones and hormone analogs; immunomodulators; photosensitizing agents;and miscellaneous other agents. Examples of these antineoplastics arelisted in Table 1.

The alkaloid class of compounds are also referred to as mitoticinhibitors, as they are cell cycle phase specific and serve to inhibitmitosis or inhibit the enzymes required for mitosis. They are derivedgenerally from plant alkaloids and other natural products and workduring the M-phase of the cell cycle. This class of compounds is oftenused to treat neoplasias such as acute lymphoblastic leukemia, Hodgkin'sand non-Hodgkin's lymphoma; neuroblastomas and cancers of the lung,breast and testes.

Alkylating agents make up a large class of chemotherapeutic agents,including of the following sub-classes, which each represent a number ofindividual drugs: alkyl sulfonates; aziridines; ethylenimines andmethylmelamines; nitrogen mustards; nitrosoureas; and others. Alkylatingagents attack neoplastic cells by directly alkylating the DNA of cellsand therefore causing the DNA to be replication incompetent. This classof compounds is commonly used to treat a variety of diseases, includingchronic leukemias, non-Hodgkin's lymphoma, Hodgkin's lymphoma, multiplemyeloma and certain lung, breast and ovarian cancers.

Nitrosoureas are often categorized as alkylating agents, and have asimilar mechanism of action, but instead of directly alkylating DNA,they inhibit DNA repair enzymes causing replication failure. Thesecompounds have the advantage of being able to cross the blood-brainbarrier and therefore can be used to treat brain tumors.

Antitumor antibiotics have antimicrobial and cytotoxic activity and alsointerfere with DNA by chemically inhibiting enzymes and mitosis or byaltering cell membranes. They are not cell cycle phase specific and arewidely used to treat a variety of cancers.

The antimetabolite class of antineoplastics interfere with the growth ofDNA and RNA and are specific to the S-phase of the cell-cycle. They canbe broken down further by type of compound, which include folic acidanalogs, purine analogs, and pyrimidine analogs. They are often employedin the treatment of chronic leukemia, breast, ovary, andgastrointestinal tumors.

There are two classes of hormones or hormone analogs used asantineoplastic agents, the corticosteroid hormones and sex hormones.While some corticosteroid hormones can both kill cancer cells and slowthe growth of tumors, and are used in the treatment of lymphoma,leukemias, etc., sex hormones function primarily to slow the growth ofbreast, prostate and endometrial cancers. There are numerous subclassesof hormones and hormone analogs, including, androgens, antiadrenals,antiandrogens, antiestrogens, aromatase inhibitors, estrogens,leutenizing hormone releasing hormone (LHRH) analogs and progestins.

An additional smaller class of antineoplastics is classified asimmunotherapy. These are agents which are intended to stimulate theimmune system to more effectively attack the neoplastic cells. Thistherapy is often used in combination with other therapies.

There are also a number of compounds, such as campothectins, which aregenerally listed as ‘other’ antineoplastic agents and can be used totreat a variety of neoplasias.

While there is a plethora of antineoplastic agents, the efficacy ofthese compounds is often outweighed by the severity of the side effectsproduced by the agent. This comparison is often referred to as thetherapeutic index, which describes the balance between the required doseto accomplish the destruction of the cancer cells compared to the doseat which the substance is unacceptably toxic to the individual. Thedrawback to most antineoplastic agents is the relatively small range ofthe therapeutic index, (i.e, the narrow dosage range in which cancercells are destroyed without unacceptable toxicity to the individual).This characteristic limits the frequency and dosage where an agent isuseful, and often the side effects become intolerable before the cancercan be fully eradicated.

The severe side effects experienced with the majority of cancerchemotherapeutics are a result of the non-specific nature of thesedrugs, which do not distinguish between healthy and cancerous cells, andinstead destroy both. The cell cycle specific drugs attempt to lessenthese effects, targeting phases of the cell cycle involved in cellreplication and division. These drugs do not, however, distinguishbetween cancerous cells and healthy cells which are undergoing normalcell division. The cells most at risk from these types of chemotherapyare those which undergo cell division often, including blood cells, hairfollicle cells, and cells of the reproductive and digestive tracts.

The most common side effects of antineoplastic agents are nausea andvomiting. A large proportion of individuals also suffer frommyelosuppression, or suppression of the bone marrow, which produces redblood cells, white blood cells and platelets. These and other sideeffects are also exacerbated by the suppression of the immune systemconcomitant with the destruction and lack of production of white bloodcells, and associated risk of opportunistic infection.

Other side effects common to a wide range of antineoplastic agentsinclude: hair loss (alopecia); appetite loss; weight loss; tastechanges; stomatitis and esophagitis (inflammation and sores);constipation; diarrhea; fatigue; heart damage; nervous system changes;lung damage; reproductive tissue damage; liver damage; kidney andurinary system damage.

The wide range of the side effects associated with most antineoplasticagents and their severity in individuals who are already debilitatedwith disease and possibly immune compromised has led researches tosearch for mechanisms by which they can alleviate some of the sideeffects while maintaining the efficacy of the treatment. Severalapproaches to this problem have been taken. They include combinationchemotherapy, where multiple antineoplastics are administered together;adjuvant therapies, where additional agents are prescribed along withthe antineoplastic agent to fight the side effects of theantineoplastic; alternative delivery vehicles for the administration ofchemotherapeutics, such as the encapsulation of antineoplastic agents inliposomes; and combined modality treatments, where chemotherapy iscombined with radiation and/or surgery.

One difficulty with respect to combination chemotherapy is that manyantineoplastic agents have similar side effects, so while their toxicityprofiles are different, the individual will still suffer greatly and maynot be able to finish the recommended course of treatment.

Another aspect of combination chemotherapy is the addition of hormonesto the combination of drugs administered. While the hormone or hormonalanalog treatment is generally not cytotoxic, hormonal manipulation helpsto prevent or slow cell division and therefore slows the growth of thetumor. This type of therapy is often used for hormone dependent tumorsof, for instance, the prostate, breast or ovaries. One well knownexample is the treatment of breast cancer with tamoxifen.

An additional method of combating the side effects associated withantineoplastics and, more importantly, extending the therapeutic dosageof these agents is adjuvant therapy, where additional agents areco-administered to the individual in order to ameliorate the sideeffects or toxicity of the antineoplastic agent. Examples of suchadjuvant therapy includes the administration of chemoprotective agents,such as the uroprotective agent mesna, the antimetastatic agentbatimastat, the folic acid replenisher folinic acid. Additionaltherapies include the administration of granulocyte colony stimulatingfactors, granulocyte-macrophage colony stimulating factor and even thetransplantation of hematopoietic stem cells. These last three therapiesaim to treat lessen the chance of opportunistic infection due tomyelosuppression concomitant with many chemotherapy regimens. However,despite the recent advances in antineoplastic and adjuvant therapy thereare still numerous cancers, for example ovarian cancer, that areresistant to current treatments, and leave the individual at risk forpotentially serious infection.

Radiation Therapy

Along with chemotherapy and surgery, radiation is one of the mostcommonly used treatment modalities, used in approximately 60% oftreatment regimens. Radiation, in any of several forms, is often used asthe primary therapy for basal cell carcinomas of the skin, head andneck, prostate cancers, bladder cancers, and others. Often combined withchemotherapy and/or surgery, radiation therapy encompasses both localand total body administration as well as a number of new advances,including radioimmunotherapy.

The cytotoxic effect of radiation on neoplastic cells arises from theability of radiation to cause a break in one or both strands of the DNAmolecule inside the cells. Cells in all phases of the cell cycle aresusceptible to this effect. However, the DNA damage is more likely to belethal in cancerous cells because they are less capable of repairing DNAdamage. Healthy cells, with functioning cell cycle check proteins andrepair enzymes, are far more likely to be able to repair the radiationdamage and function normally after treatment.

Tumors and tissues themselves are also characterized by a range ofsusceptibilities to radiation therapy. Lymphoma and leukemias are verysensitive to radiation therapy, while renal cancer and gland tumors arefairly insensitive to radiation. A tumor that is consideredradiosensitive is one which can be eradicated by a dose(s) of radiationthat is also well tolerated by the surrounding tissues. Unsurprisingly,different tissue types within the body tolerate radiation at differentdoses. Tissues that undergo frequent cell division are most effected bytreatment, similar to their sensitivity to certain cell cycle specificchemotherapy agents.

The radiosensitivity of tumors is also effected by hypoxia, or a lack ofoxygen in the interiors of larger tumors. Hypoxic tumors can be 2-3times less responsive to radiation treatment. Certain agents used inconjunction with radiation treatment, such as some of theradiosensitizing agents, work by increasing the singlet oxygen speciesin the vicinity of the tumor and therefore increasing itsradiosensitivity. Other compounds used in conjunction with radiationtherapy include radioprotectants which are designed to protectsurrounding tissue from some of the effects of radiation therapy.Sources of radiation include: Americium, chromic phosphate, radioactive,Cobalt, ¹³¹I-ethiodized oil, Gold (radioactive, colloidal) iobenguane,Radium, Radon, sodium iodide (radioactive), sodium phosphate(radioactive).

Radiation therapy itself can be classified according to two primarytypes, internal and external radiation therapy. External therapyinvolves the administration of radiation via a machine capable ofproducing high-energy external beam radiation. This therapy can includeeither total body irradiation, or can be localized to the region of thetumor. With external radiation treatments, the bodily secretions of theindividual are not radioactive after treatment. The radiation itself canbe either electromagnetic (X-ray or gamma radiation) or particulate (αor β particles). The treatment requirements will differ depending uponthe characteristics of the tumor. External radiation is often used pre-or post-operatively; either to shrink the tumor before surgery, or tomop up remaining cancer cells after surgery.

Internal radiation therapy, also termed brachytherapy, involvesimplantation of a radioactive isotope as the source of the radiation.There a variety of methods of delivery, including permanent, temporary,sealed, unsealed, intracavity or interstitial implants. The choice ofimplant is determined by a variety of factors, including the locationand extent of the tumor.

A third, but still experimental, type of radiation therapy is oftentermed radioimmunotherapy. This involves the attachment of radioisotopesto monoclonal antibodies specific for the tumor cells. Uponadministration the antibodies specifically seek out and destroy thecancer cells.

The side effects of radiation are similar to those of chemotherapy andarise for the same reason, the damage of healthy tissue. Radiation isusually more localized than chemotherapy, but treatment is stillaccompanied by damage to previously healthy tissue. Many of the sideeffects are unpleasant, and radiation also shares with chemotherapy thedisadvantage of being mutagenic, carcinogenic and teratogenic in its ownright. While normal cells usually begin to recover from treatment withintwo hours of treatment, mutations may be induced in the genes of thehealthy cells. These risks are elevated in certain tissues, such asthose in the reproductive system. It has also been found that peopletolerate radiation differently. Doses that may not lead to new cancersin one individual may in fact spawn additional cancers in anotherindividual. This could be due to pre-existing mutations in cell cyclecheck proteins or repair enzymes, but current practice would not be ableto predict at what dose a particular individual is at risk. Common sideeffects of radiation include: bladder irritation; fatigue; diarrhea; lowblood counts; mouth irritation; taste alteration, loss of appetite;alopecia; skin irritation; change in pulmonary function; enteritis;sleep disorders; and others.

Adenovirus Vectors

Until relatively recently, the virtually exclusive focus in developmentof adenoviral vectors for gene therapy has been use of adenovirus merelyas a vehicle for introducing the gene of interest, not as an effector initself. Replication of adenovirus had previously been viewed as anundesirable result, largely due to the host immune response. Morerecently, however, the use of adenovirus vectors as effectors has beendescribed. International Patent Application Nos. PCT/US98/04084,PCT/US98/04080; PCT/US98/04133, PCT/US98/04132, PCT/US98/16312,PCT/US95/00845, PCT/US96/10838, PCT/EP98/07380, U.S. Pat. No. 5,998,205and U.S. Pat. No. 5,698,443. The use of IRES in vectors have beendescribed. See, for example, International Patent Application No.PCT/US98/03699 and International Patent Application No. PCT/EP98/07380.Adenovirus E1A and E1B genes are disclosed in Rao et al. (1992, Proc.Natl. Acad. Sci. USA vol. 89: 7742-7746).

Publications describing various aspects of adenovirus biology and/ortechniques relating to adenovirus include the following. PCT/US95/14461;Graham and Van de Eb (1973) Virology 52:456-467; Takiff et al. (1981)Lancet ii:832-834; Berkner and Sharp (1983) Nucleic Acid Research6003-6020; Graham (1984) EMBO J 3:2917-2922; Bett et al. (1993) J.Virology 67:5911-5921; and Bett et al. (1994) Proc. Natl. Acad. Sci. USA91:8802-8806 describe adenoviruses that have been genetically modifiedto produce replication-defective gene transfer vehicles. In thesevehicles, the early adenovirus gene products E1A and E1B are deleted andprovided in trans by the packaging cell line 293 developed by FrankGraham (Graham et al. (1987) J. Gen. Birol. 36:59-72 and Graham (1977)J. Genetic Virology 68:937-940). The gene to be transduced is commonlyinserted into adenovirus in the deleted E1A and E1B region of the virusgenome Bett et al. (1994), supra. Adenovirus vectors as vehicles forefficient transduction of genes have been described byStratford-Perricaudet (1990) Human Gene Therapy 1:2-256; Rosenfeld(1991) Science 252:431434; Wang et al. (1991) Adv. Exp. Med. Biol.309:61-66; Jaffe et al. (1992) Nat Gent. 1:372-378; Quantin et al.(1992) Proc Natl. Acad. Sci. USA 89:2581-2584; Rosenfeld et al. (1992)Cell 68:143-155; Stratford-Perricaudet et al. (1992) J. Clin. Invest.90:626-630; Le Gal La Salle et al. (1993) Science 259:988-990;Mastrangeli et al. (1993) J. Clin. Invest. 91:225-234; Ragot et al.(1993) Nature 361:647-650; Hayaski et al. (1994) J. Biol. Chem.269:23872-23875.

There are several other experimental cancer therapies which utilizevarious aspects of adenovirus or adenovirus vectors. See, U.S. Pat. No.5,776,743; U.S. Pat. No. 5,846,945; U.S. Pat. No. 5,801,029;PCT/US99/08592; U.S. Pat. No. 5,747,469; PCT/US98/03514; andPCT/US97/22036.

Of particular interest is the development of more specific, targetedforms of cancer therapy, especially in cancers that are difficult totreat successfully, such as prostate, bladder or ovarian cancer. Incontrast to conventional cancer therapies, which result in relativelynon-specific and often serious toxicity, more specific treatmentmodalities attempt to inhibit or kill malignant cells selectively whileleaving healthy cells intact. There is, therefore a serious need fordeveloping specific, less toxic cancer therapies.

All references cited herein are hereby incorporated by reference intheir entirety.

SUMMARY OF THE INVENTION

The invention provides methods for the administration of combinations ofa target cell-specific adenoviral vector and at least one antineoplasticagent(s) and/or radiation to an individual in need thereof, such as, anindividual with neoplasia.

Accordingly, in one aspect, the invention provides methods ofsuppressing tumor growth in an individual comprising the steps of: a)administering to the individual a composition comprising areplication-competent target cell-specific adenoviral vector whereinsaid vector comprises an adenovirus gene essential for replication(preferably an early gene) under transcriptional control of a targetcell specific transcriptional regulatory element (TRE); and b)administering an antineoplastic agent to the individual, wherein theadenoviral vector and antineoplastic agent are administered in amountssufficient to suppress tumor growth. In some embodiments, the amount ofadenovirus vector and/or anitneoplastic agent administered is less thanthat known in the art to be effective for suppressing tumor growth wheneither is administered alone. In one embodiment, the antineoplasticagent includes alkaloids, alkylating agents, antibiotics,antimetabolites, immunomodulators, nitrosoureas, hormoneantagonists/agonists and analogs, or photosensitizing agents.

In another aspect, the invention provides methods of suppressing tumorgrowth in an individual comprising the following steps: a) administeringto the individual a composition comprising a replication-competenttarget cell-specific adenoviral vector wherein said vector comprises anadenovirus gene essential for replication (preferably an early gene)under transcriptional control of a target cell specific transcriptionalregulatory element (TRE); and b) administering an effective amount ofradiation. In some embodiments, the amount of adenovirus vector and/orradiation administered is less than that known in the art to beeffective for suppressing tumor growth when administered alone. In oneembodiment, the radiation includes X-rays, gamma rays, alpha particles,beta particles, electrons, photons, neutrons, other ionizing radiationor radioactive isotopes.

In yet another aspect, the present invention provides methods forsuppressing tumor growth in an individual comprising the followingsteps, in any order: a) administering to the individual an effectiveamount of a replication-competent target cell-specific adenoviral vectorand an effective amount of at least one antineoplastic agent; and b)administering an effective amount of an appropriate course of radiationtherapy to the individual. In one embodiment, the method may furthercomprise, c) administering to the individual an additional dose of theadenoviral/chemotherapeutic solution or radiation as necessary to treatthe individual's neoplasia In another embodiment, the method may furthercomprise a delay between any of steps a), b) and c). In someembodiments, the amount of adenovirus vector and/or anitneoplastic agentand/or radiation administered will be less than that known in the art tobe effective for suppressing tumor growth when either is administeredalone.

Any TRE which directs cell-specific expression can be used in thedisclosed adenovirus vectors. In one embodiment, TREs include, forexample, TREs specific for prostate cancer cells, breast cancer cells,hepatoma cells, melanoma cells, bladder cells or colorectal cancercells. In another embodiment, the TREs include, probasin (PB) TRE;prostate-specific antigen (PSA) TRE; mucin (MUC1) TRE; α-fetoprotein(AFP) TRE; hKLK2 TRE; tyrosinase TRE; human uroplakin II TRE (hUPII) orcarcinoembryonic antigen (CEA) TRE. In other embodiments, the targetcell-specific TRE is a cell status-specific TRE. In yet otherembodiments, the target cell-specific TRE is a tissue specific TRE.

In one aspect, the adenovirus vectors comprise adenovirus genesessential for viral replication. An essential gene can be an early viralgene, including for example, E1A; E1B; E2; and/or E4, or a late viralgene. In another aspect, the adenovirus vector comprises E3.

In some embodiments, the adenovirus vectors comprise an adenovirus genehaving an inactivation of its endogenous promoter. In one embodiment,the adenovirus gene is essential for viral replication under control ofa target cell-specific TRE. In another embodiment, the adenovirus geneis E1A wherein the E1A promoter is inactivated and wherein the E1A geneis under transcriptional control of a heterologous cell-specific TRE. Inanother embodiment, the adenovirus gene is E1B wherein the E1B promoteris inactivated and wherein the E1B gene is under transcriptional controlof a heterologous cell-specific TRE. In other embodiments, theadenovirus vectors comprise the adenovirus gene, E1B, having a deletionof the 19-kDa region.

In other embodiments, an enhancer element for the adenovirus genes isinactivated, such as an inactivation of E1A enhancer. In yet otherembodiments, the E1A promoter is inactivated and the E1A enhancer I isinactivated. In further embodiments, the TRE has its endogenous silencerelement inactivated.

In another embodiment, the replication competent adenovirus vectorscomprise co-transcribed first and second genes under transcriptionalcontrol of a heterologous, target cell-specific transcriptionalregulatory element (TRE), wherein the second gene is under translationalcontrol of an internal ribosome entry site (IRES). In one aspect, thefirst and/or second genes are adenovirus genes and in another aspect,the first and/or second adenovirus genes are essential for viralreplication. An essential gene can be an early viral gene, including forexample, E1A; E1B; E2; and/or E4, or a late viral gene. In anotheraspect an early gene is E3.

In one embodiment, the first gene is an adenovirus gene and the secondgene is a therapeutic gene. In another embodiment, both genes areadenovirus genes. In an additional embodiment, the first adenovirus geneis E1A, and the second adenovirus gene is E1B. Optionally, theendogenous promoter for one of the co-transcribed adenovirus geneessential for viral replication, such as for example, E1A, isinactivated, placing the gene under sole transcriptional control of atarget cell-specific TRE.

In additional embodiments, the adenovirus vector comprises at least oneadditional co-transcribed gene under the control of the cell-specificTRE. In another embodiment, an additional co-transcribed gene is underthe translational control of an IRES.

In another aspect of the present invention, adenovirus vectors furthercomprise a transgene such as, for example, a cytotoxic gene. In oneembodiment, the transgene is under the transcriptional control of thesame TRE as the first gene and second genes and optionally under thetranslational control of an internal ribosome entry site. In anotherembodiment, the transgene is under the transcriptional control of adifferent TRE that is functional in the same cell as the TRE regulatingtranscription of the first and second genes and optionally under thetranslational-control of an IRES.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B is a schematic depicting target cell-specific adenovirusvectors described in the Examples.

FIG. 2 is a graph depicting percent viable LNCaP prostate tumor cellstreated with CV787 adenovirus vector (solid circles; MOI 0.01); CV787and TAXOL™ (paclitaxel; solid squares); TAXOL™ alone (solid triangles;6.25 nM) and mock infected control (diamonds). For the combinedadministration of CV787 and TAXOL™, TAXOL™ was administered first, 24hrs prior to CV787.

FIG. 3 is a graph depicting percent viable LNCaP prostate tumor cellstreated with CV787 adenovirus vector (solid circles; MOI 0.01); CV787and TAXOTERE™ (docetaxel; solid squares); TAXOTERE™ alone (triangles;3.12 nM); and mock infected control (diamonds) In the combinationadministration, TAXOTERE™ was administered first.

FIG. 4 is a graph depicting percent viable LNCaP prostate tumor cellstreated with CV787 adenovirus vector (solid circles; MOI 0.01); CV787and TAXOTERE™ (docetaxel; solid squares); TAXOTERE™ alone (triangles;3.12 nM); and mock infected control (diamonds) For the combinationadministration, CV787 was added first.

FIG. 5 is a graph depicting percent viable LNCaP prostate tumor cellstreated with CV787 adenovirus vector (solid circles; MOI 0.1); CV787 andmitoxantrone (MTX; solid squares); MTX alone (solid circles with “X”;100 nM); and mock infected control (diamonds). For the combinationadministration, Mitoxantrone was administered first, 24 hrs prior toCV787.

FIG. 6 is a bar graph depicting percent viable LNCaP prostate tumorcells with no treatment (mock); CV787 treatment (MOI 0.01); etoposidetreatment (500 ng/ml); and CV787 plus etoposide (Eto) treatment on day 8(Etoposide was administered first).

FIG. 7 is a bar graph depicting percent viable LNCaP prostate tumorcells with no treatment; CV787 treatment (MOI 0.01); doxorubicintreatment (50 ng/ml); and CV787 plus doxorubicin (Doxo) treatment on day8 (CV787 was administered first).

FIG. 8 is a bar graph depicting percent viable LNCaP prostate tumorcells with no treatment; CV787 treatment (MOI 0.1); cisplatin treatment(8.25 μM); and CV787 plus cisplatin (Cis) treatment on day 5 (Cisplatinwas administered first).

FIG. 9 is a bar graph depicting percent viable LNCaP prostate tumorcells with no treatment; CV787 treatment (MOI 0.01); 5-fluorouracil(5-FU; 35 μM) treatment; and CV787 plus 5-fluorouracil treatment on day8 (5-fluorouracil was administered first).

FIG. 10 is a graph depicting percent viable LNCaP prostate tumor cellstreated with CV787 adenovirus vector (solid circles; MOI 0.01); CV787and radiation (solid squares); radiation alone (solid triangles; ¹³⁷Cs;2 Gy); and mock infected control (diamonds). For combinationadministration, CV787 was administered first, 24 hours prior toradiation.

FIG. 11 is a graph depicting CV787 adenovirus yield in LNCaP prostatetumor cells treated with CV787 (MOI 0.1) and mock infected control;CV787 and TAXOL™ (6.25 nM); CV787 and mitoxantrone (Mito; 100 nM); CV787and doxorubicin (Dox; 50 ng/ml); and CV787 and etoposide (500 ng/ml), onday 6 of treatment. For all combination administration, CV787 wasadministered first.

FIG. 12 is a bar graph depicting CV787 adenovirus yield in LNCaPprostate tumor cells (dashed shading); HBL-100 breast epithelial cells(horizontal shading); and PA-1 ovary cells (solid shading) when treatedwith CV787 adenovirus vector (MOI 0.1); CV787 and TAXOL™ (6.25 nM);CV787 and mitoxantrone (MTX; 100 nM); and CV787 and doxorubicin (Doxo;50 ng/ml). For combination administration, CV787 was administered firstwith virus yield measured at 72 hours after infection.

FIG. 13 is a graph depicting relative percent viable cells forcombination treatment compared to chemotherapeutic agent alone over timewhen treated with CV787 adenovirus vector (MOI 0.01) and TAXOL™ (6.25nM). LNCaP, prostate tumor cells (solid circles); HBL-100, breastepithelial tissue cells (solid triangles); OVCAR-3, ovarian cancer cells(solid diamonds); and 293, human embryonic kidney cells (solid squares),E1A and E1B permissible. For combination administration, CV787 wasadministered first.

FIG. 14 is a bar graph depicting percent viable cells when treated withCV787 adenovirus vector (dark shading; MOI 0.1); CV787 and mitoxantrone(MTX; outlined; 100 nM) and mitoxantrone alone (horizontal shading) onday 7 of treatment. LNCaP, prostate tumor cells; HBL-100, breastepithelial tissue cells; OVCAR-3, ovarian cancer cells; and 293, humanembryonic kidney cells, E1A and E1B permissible. For combinationadministration, CV787 was administered first.

FIG. 15 is a graph depicting percent viable Hep3B (3B) and HepG2 (G2)hepatoma cells treated with CV790 adenovirus vector (diamonds; MOI0.01); CV790 and doxorubicin (triangles); and doxorubicin alone(squares; 10 ng/ml). For combination administration, CV790 wasadministered first.

FIG. 16 is a graph depicting percent viable HepG2 (G2) hepatoma cellstreated with CV790 adenovirus vector (solid diamonds; MOI 0.01); CV790and doxorubicin (solid triangles); and doxorubicin alone (solid squares;10 ng/ml). For combination administration, Doxorubicin was administeredfirst.

FIG. 17 is a graph depicting percent viable HepG2 (G2) hepatoma cellstreated with CV790 adenovirus vector (solid diamonds; MOI 0.01); CV790and doxorubicin (solid triangles); and doxorubicin alone (solid squares;10 ng/ml). For combination administration, CV790 and doxorubicin wereadministered together.

FIG. 18 is a graph depicting percent viable HepG2 (G2) and Hep3B (3B)hepatoma cells treated with CV790 adenovirus vector (diamonds; MOI 0.1);CV790 and cisplatin (triangles); and cisplatin alone (squares; 1 μg/ml).For combination administration, CV790 was administered first.

FIG. 19 is a graph depicting percent viable HepG2 (G2) and Hep3B (3B)hepatoma cells treated with CV790 adenovirus vector (diamonds; MOI 0.1);CV790 and TAXOL™ (paclitaxel; triangles); and TAXOL™ alone (squares; 0.5ng/ml). For combination administration, CV790 was administered first.

FIG. 20 is a graph depicting percent viable HepG2 (G2) and Hep3B (3B)hepatoma cells treated with CV790 adenovirus vector (diamonds; MOI 0.1);CV790 and 5-fluorouracil (triangles); and 5-fluorouracil alone (squares;10 ng/ml). For combination administration, CV790 was administered first.

FIG. 21 is a graph depicting percent viable HepG2 (G2) and Hep3B (3B)hepatoma cells treated with CV790 adenovirus vector (diamonds; MOI 0.1);CV790 and mitoxantrone (triangles); and mitoxantrone alone (squares; 4ng/ml). For combination administration, CV790 was administered first.

FIG. 22 is a graph depicting percent viable HepG2 (G2) and Hep3B (3B)hepatoma cells treated with CV790 adenovirus vector (diamonds; MOI 0.1);CV790 and mitomycin C (triangles); and mitomycin C alone (squares; 10ng/ml). For combination administration, CV790 was administered first.

FIG. 23 is a graph depicting the tumor volume of LNCaP prostate tumorxenografts treated with CV787 adenovirus vector (triangles; 1×10⁷particles/mm³); CV787 and TAXOL™ (solid squares); TAXOL™ alone(paclitaxel; solid circles; 15 mg/kg); and mock infected control (soliddiamonds). For combination administration, CV787 was administered firston day 0 via intra-tumor injection. TAXOL™ was administered on day 1, 2,3, and 4.

FIG. 24 is a graph depicting the relative percent of tumor volume ofLNCaP prostate tumor xenografts treated with TAXOL™ and CV787 adenovirusvector (triangles; TAXOL™ 2 mg/kg; 1×10¹⁰ particles); CV787 and TAXOL™(solid squares; TAXOL™ 10 mg/kg); TAXOL™ alone (solid circles; 10mg/kg); and TAXOL™ alone (solid diamonds; 2 mg/kg). For combinationadministration, TAXOL™ was administered first via intravenousadministration.

FIG. 25 is a graph depicting the tumor volume of LNCaP prostate tumorxenografts treated with CV787 adenovirus vector (triangles; 1×10¹⁰particles); CV787 and TAXOL™ (solid squares); TAXOL™ alone (solidcircles; 20 mg/kg); mock infected control (vehicle; solid diamonds). Forcombination administration, CV787 was administered first via intravenousdelivery.

FIG. 26 is a graph depicting the relative percent tumor volume of LNCaPprostate tumor xenografts treated with CV787 adenovirus vector (shadedsquares; 1×10¹¹ particles); CV787 (solid circles; 1×10¹⁰ particles);CV787 (1×10¹⁰ particles) and mitoxantrone (Mito; “X”; 3 mg/kg); CV787(1×10¹¹ particles) and mitoxantrone (solid diamonds; 3 mg/kg);mitoxantrone alone (solid triangles; 3 mg/kg) and mock infected control(vehicle; “-”). For combination administration, CV787 was administeredfirst.

FIG. 27 is a graph depicting the relative percent tumor volume of LNCaPprostate tumor xenografts treated with CV787 adenovirus vector (solidcircles; 1×10¹⁰ particles); CV787 and estramustine (solid squares);estramustine alone (triangles); and mock infected control (soliddiamonds). For combination administration, CV787 was administered first.Estramustine was administered at 14 mg/kg on days 2-5, 7-11, 13-17 and20-24.

FIG. 28 is a graph depicting the tumor volume of LNCaP prostate tumorxenografts treated with CV787 adenovirus vector (solid circles; 1×10¹⁰particles), CV787 and docetaxel (solid squares; 1×10¹⁰ particles, 10mg/kg); docetaxel alone (solid triangles; 10 mg/kg); and mock infectedcontrol (shaded diamonds). For combination administration, CV787 wasadministered first.

FIG. 29 is a graph depicting the tumor volume of LNCaP prostate tumorxenografts treated with CV787 adenovirus vector (shaded triangles;1×10¹⁰ particles), CV787 (unfilled triangles; 1×10¹¹ particles); CV787and docetaxel (solid squares; 1×10¹⁰ particles, 5 mg/kg); docetaxelalone (solid circles; 5 mg/kg); and mock infected control (soliddiamonds). For combination administration, CV787 was administered first.

FIG. 30 is a graph depicting the relative percent tumor volume of Hep3Bhepatoma xenografts treated with CV790 adenovirus vector (solid circles;1×10¹¹ particles); CV790 and doxorubicin (Doxo; solid squares);doxorubicin alone (triangles; 10 mg/kg); and mock infected control(solid diamonds). For combination administration, CV790 was administeredfirst.

FIG. 31 is a graph depicting the relative percent tumor volume of Hep3Bhepatoma xenografts treated with CV890 adenovirus vector (solid circles;1×10¹¹ particles); CV890 and doxorubicin (solid squares); doxorubicinalone (triangles; 10 mg/kg); and mock infected control (solid diamonds).For combination administration, CV890 was administered first.

FIG. 32 is a graph depicting percent viable LNCaP prostate tumor cellstreated with CV787 adenovirus vector (solid circles; MOI 0.1); CV787 andradiation (solid squares); radiation alone (solid triangles; 6 Gy); andno treatment (diamonds). In combination administration, radiation wasadministered first.

FIG. 33 is a graph depicting percent viable LNCaP prostate tumor cellstreated with CV787 adenovirus vector (solid circles; MOI 0.1); CV787 andradiation (solid squares); radiation alone (solid triangles; 6 Gy); andno treatment (diamonds). In combination administration, CV787 wasadministered first.

FIG. 34 is a graph depicting the virus yield of CV787 adenovirus vectorover time for CV787 administered with radiation first (solid squares;MOI 0.1; 6 Gy) and CV787 administered without radiation (solid circles).

FIG. 35 is a graph depicting the virus yield of CV787 adenovirus vectorover time for CV787 administered before radiation (solid squares; MOI0.1; 6 Gy) and CV787 administered without radiation (solid circles).

FIG. 36 is a graph depicting percent of cell death of LNCaP prostatetumor cells treated with CV787 adenovirus vector (MOI 0.01) andincreasing doses of radiation, on day 6 of treatment. CV787 wasadministered first.

FIG. 37 depicts a nucleotide and amino acid sequence for ADP.

FIG. 38 depicts an IC₅₀ isobologram of doxorubicin and CV 890 on Hep3Bcells at day 5.

FIG. 39 depicts in vivo efficacy of CV890 with doxorubicin. Hep3B nudemouse xenografts were grouped (n=6) and treated with CV890 alone (1×10¹¹particles/dose, iv), doxorubicin alone (10 mg/kg, ip), CV890 anddoxorubicin combination (1×10¹¹ particles of CV890 through tail vein and10 mg/kg doxorubicin ip), or vehicle control. Tumor size was measuredweekly and the tumor volume were normalized as 100% at the day oftreatment. Error bars represent the standard error of the mean.

FIG. 40 shows the virus yield of CV802, CV882 and CV884 in cell lines.

FIG. 41 are schematic depictions of various adenovirus constructsdescribed herein.

MODES FOR CARRYING OUT THE INVENTION

We have discovered methods of using replication-competent, targetcell-specific adenovirus vectors in combination with singlechemotherapeutic agents, combinations of chemotherapeutic agents,radiation therapy treatment and the combination of radiation therapy andchemotherapeutic agents. The target cell-specific replication-competentadenovirus vectors comprise an adenovirus gene essential forreplication, preferably an early gene, under the transcriptional controlof a cell type-specific transcriptional regulatory element (TRE). Byproviding for cell type-specific transcription through the use of one ormore cell type-specific TREs, the adenovirus vectors effectcell-specific cytotoxicity due to selective replication. We haveobserved synergy with respect to these adenoviral vectors and variouschemotherapeutic agents as well as radiation compared to results usingadenovirus or chemotherapy or radiation alone.

Although chemotherapeutic agents are used to treat a wide variety ofcancers, the success rate is highly variable and the chemotherapeuticagents themselves are highly toxic, causing highly undesirable sideeffects and possibly contributing additional mutagenic or carcinogenicresults in an already immune-compromised individual. Because thecombination of adenoviral vectors and chemotherapeutics cansynergistically enhance the efficacy of treatment, this in turn permitsa lower effective dose of virus and/or chemotherapeutic agent, reducingthe toxicity of the treatment and the suffering of the individual. Anadditional potential benefit is reduced length of treatment, as we haveobserved that tumors respond to the combined viral therapy more quicklythan to chemotherapy or viral therapy alone.

We have also discovered that, in spite of their potential to damageviral DNA and thus compromise adenoviral vector function, viralreplication is not appreciably changed in the presence ofchemotherapeutic agent(s) and/or radiation, and that simultaneousadministration of target-cell specific adenovirus and chemotherapeuticagent(s) is effective for killing tumor cells.

In some embodiments, the methods are for suppressing tumor growth. Inother embodiments, the methods are for reducing size and/or extent of atumor. In other embodiments, the methods are for delaying development ofa tumor. In other embodiments, the methods are for treating a neoplasiaIn still other embodiments, the methods are for killing tumor cells.

With respect to all methods described herein, target cells (i.e.,neoplastic, proliferative cells) are contacted with an appropriateadenovirus vector described herein (preferably in the form of anadenovirus particle) such that the vector enters the cell and viralreplication initiates. Target cell(s) are also contacted with anotheragent which kills tumor cells, such as a chemotherapeutic agent(s)and/or radiation.

Individuals suitable for treatment by these methods include individualswho have or are suspected of having neoplasia, including individuals inthe early or late stages of the disease, as well as individuals who havepreviously been treated (e.g., are in the adjuvant setting). Otherindividuals suitable for the methods described herein are those who areconsidered high risk for developing a tumor, such as those who have agenetic predisposition to development of a neoplasia and/or who havebeen exposed to an agent(s) which is correlated with development of aneoplasia. Treatment regimes include both the eradication of tumors orother forms of the disease as well as palliation of the disease. Thesemethods of treatment are suitable for numerous forms of neoplasia,including, but not limited to bladder cancer, prostate cancer, livercancer, breast cancer, colon cancer, melanoma, ovarian pancreatic, lung,and brain cancer.

The presence of neoplasia and the suitability of the individual forreceiving the methods described herein may be determined by any of thetechniques known in the art, including diagnostic methods such asimaging techniques, analysis of serum tumor markers, and biopsy.

The various methods of the invention will be described below. Certainembodiments of the methods use replication-competent targetcell-specific adenoviral vectors such as CV706 (prostate specific);CV787 (prostate specific); CV790 (liver specific); CV829 (bladderspecific); CV884 (bladder specific); CV859 (melanoma specific); CV873(colon/breast specific); CV890 (liver specific); CV874 (bladderspecific); CV875 (bladder specific); CV876 (bladder specific); CV877(bladder specific) and CV855 (melanoma specific), as described herein. Asummary of the components of these vectors is included in the Examplessection as Table 4. Although methods of tumor suppression areexemplified in the discussion below, it is understood that thealternative methods described above are equally applicable and suitablefor these methods, and that the endpoints of these methods are measuredusing methods standard in the art, including the diagnostic andassessment methods described above.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the scope of those of skill in the art.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook etal., 1989); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “AnimalCell Culture” (R. I. Freshney, ed., 1987); “Methods in Enzymology”(Academic Press, Inc.); “Handbook of Experimental Immunology” (D. M.Weir & C. C. Blackwell, eds.); “Gene Transfer Vectors for MammalianCells” (J. M. Miller & M. P. Calos, eds., 1987); “Current Protocols inMolecular Biology” (F. M. Ausubel et al., eds., 1987); “PCR: ThePolymerase Chain Reaction”, (Mullis et al., eds., 1994); and “CurrentProtocols in Immunology” (J. E. Coligan et al., eds., 1991).

For techniques related to adenovirus, see, inter alia, Felgner andRingold (1989) Nature 337:387-388; Berkner and Sharp (1983) Nucl. AcidsRes. 11:6003-6020; Graham (1984) EMBO J. 3:2917-2922; Bett et al. (1993)J. Virology 67:5911-5921; Bett et al. (1994) Proc. Natl. Acad. Sci. USA91:8802-8806.

Definitions

As used herein, the terms “neoplastic cells”, “neoplasia”, “tumor”,“tumor cells”, “cancer” and “cancer cells”, (used interchangeably) referto cells which exhibit relatively autonomous growth, so that theyexhibit an aberrant growth phenotype characterized by a significant lossof control of cell proliferation. Neoplastic cells can be malignant orbenign.

The terms “antineoplastic agent”, “antineoplastic chemotherapeuticagent”, “chemotherapeutic agent”, “antineoplastic” and“chemotherapeutic” are used interchangeably herein and refer to chemicalcompounds or drugs which are used in the treatment of cancer e.g., tokill cancer cells and/or lessen the spread of the disease.

“Radiation therapy” is a term commonly used in the art to refer tomultiple types of radiation therapy including internal and externalradiation therapy, radioimmunotherapy, and the use of various types ofradiation including X-rays, gamma rays, alpha particles, beta particles,photons, electrons, neutrons, radioisotopes, and other forms of ionizingradiation. As used herein, the term “radiation therapy” is inclusive ofall of these types of radiation therapy, unless otherwise specified.

As used herein, “suppressing tumor growth” refers to reducing the rateof growth of a tumor, halting tumor growth completely, causing aregression in the size of an existing tumor, eradicating an existingtumor and/or preventing the occurrence of additional tumors upontreatment with the compositions, kits or methods of the presentinvention. “Suppressing” tumor growth indicates a growth state that iscurtailed when compared to growth without contact with, i.e.,transfection by, an adenoviral vector combined with administration ofchemotherapeutic agents and radiation as described herein. Tumor cellgrowth can be assessed by any means known in the art, including, but notlimited to, measuring tumor size, determining whether tumor cells areproliferating using a ³H-thymidine incorporation assay, or countingtumor cells. “Suppressing” tumor cell growth means any or all of thefollowing states: slowing, delaying, and stopping tumor growth, as wellas tumor shrinkage.

“Delaying development” of a tumor means to defer, hinder, slow, retard,stabilize, and/or postpone development of the disease. This delay can beof varying lengths of time, depending on the history of the diseaseand/or individual being treated.

As used herein, “synergy” or “synergistic effect” when referring tocombination administration of adenovirus vector and antineoplastic agentand/or radiation means that the effect of the combination is more thanadditive when compared to administration of adenovirus vector,antineoplastic agent or radiation alone.

An “adenovirus vector” or “adenoviral vector” (used interchangeably)comprises a polynucleotide construct of the invention. A polynucleotideconstruct of this invention may be in any of several forms, including,but not limited to, DNA, DNA encapsulated in an adenovirus coat, DNApackaged in another viral or viral-like form (such as herpes simplex,and AAV), DNA encapsulated in liposomes, DNA complexed with polylysine,complexed with synthetic polycationic molecules, conjugated withtransferrin, and complexed with compounds such as PEG to immunologically“mask” the molecule and/or increase half-life, and conjugated to anonviral protein. Preferably, the polynucleotide is DNA. As used herein,“DNA” includes not only bases A, T, C, and G, but also includes any oftheir analogs or modified forms of these bases, such as methylatednucleotides, internucleotide modifications such as uncharged linkagesand thioates, use of sugar analogs, and modified and/or alternativebackbone structures, such as polyamides. For purposes of this invention,adenovirus vectors are replication-competent in a target cell.

As used herein, a “transcription response element” or “transcriptionalregulatory element”, or “TRE” is a polynucleotide sequence, preferably aDNA sequence, which increases transcription of an operably linkedpolynucleotide sequence in a host cell that allows that TRE to function.A TRE can comprise an enhancer and/or a promoter. A “transcriptionalregulatory sequence” is a TRE. A “target cell-specific transcriptionalresponse element” or “target cell-specific TRE” is a polynucleotidesequence, preferably a DNA sequence, which is preferentially functionalin a specific type of cell, that is, a target cell. Accordingly, atarget cell-specific TRE transcribes an operably linked polynucleotidesequence in a target cell that allows the target cell-specific TRE tofunction. The term “target cell-specific”, as used herein, is intendedto include cell type specificity, tissue specificity, developmentalstage specificity, and tumor specificity, as well as specificity for acancerous state of a given target cell. “Target cell-specific TRE”includes cell type-specific and cell status-specific TRE, as well as“composite” TREs. The term “composite TRE” includes a TRE whichcomprises both a cell type-specific and a cell status-specific TRE. Atarget cell-specific TRE can also include a heterologous component,including, for example, an SV40 or a cytomegalovirus (CMV) promoter(s).An example of a target cell specific TRE which is tissue specific is aCMV TRE which contains both promoter(s) and enhancer(s).

As described in more detail herein, a target cell-specific TRE cancomprise any number of configurations, including, but not limited to, atarget cell-specific promoter; and target cell-specific enhancer; aheterologous promoter and a target cell-specific enhancer; a targetcell-specific promoter and a heterologous enhancer; a heterologouspromoter and a heterologous enhancer; and multimers of the foregoing.The promoter and enhancer components of a target cell-specific TRE maybe in any orientation and/or distance from the coding sequence ofinterest, as long as the desired target cell-specific transcriptionalactivity is obtained. Transcriptional activation can be measured in anumber of ways known in the art (and described in more detail below),but is generally measured by detection and/or quantitation of mRNA orthe protein product of the coding sequence under control of (i.e.,operably linked to) the target cell-specific TRE. As discussed herein, atarget cell-specific TRE can be of varying lengths, and of varyingsequence composition. As used herein, the term “cell status-specificTRE” is preferentially functional, i.e., confers transcriptionalactivation on an operably linked polynucleotide in a cell which allows acell status-specific TRE to function, i.e., a cell which exhibits aparticular physiological condition, including, but not limited to, anaberrant physiological state. “Cell status” thus refers to a given, orparticular, physiological state (or condition) of a cell, which isreversible and/or progressive. The physiological state may be generatedinternally or externally; for example, it may be a metabolic state (suchas in response to conditions of low oxygen), or it may be generated dueto heat or ionizing radiation. “Cell status” is distinct from a “celltype”, which relates to a differentiation state of a cell, which undernormal conditions is irreversible. Generally (but not necessarily), asdiscussed herein, a cell status is embodied in an aberrant physiologicalstate, examples of which are given below.

A “functional portion” of a target cell-specific TRE is one whichconfers target cell-specific transcription on an operably linked gene orcoding region, such that the operably linked gene or coding region ispreferentially expressed in the target cells.

By “transcriptional activation” or an “increase in transcription,” it isintended that transcription is increased above basal levels in thetarget cell (i.e., target cell) by at least about 2 fold, preferably atleast about 5 fold, preferably at least about 10 fold, more preferablyat least about 20 fold, more preferably at least about 50 fold, morepreferably at least about 100 fold, more preferably at least about 200fold, even more preferably at least about 400 fold to about 500 fold,even more preferably at least about 1000 fold. Basal levels aregenerally the level of activity (if any) in a non-target cell (i.e., adifferent cell type), or the level of activity (if any) of a reporterconstruct lacking a target cell-specific TRE as tested in a target cellline.

A “functionally-preserved variant” of a target cell-specific TRE is atarget cell-specific TRE which differs from another target cell-specificTRE, but still retains target cell-specific transcription activity,although the degree of activation may be altered (as discussed below).The difference in a target cell-specific TRE can be due to differencesin linear sequence, arising from, for example, single base mutation(s),addition(s), deletion(s), and/or modification(s) of the bases. Thedifference can also arise from changes in the sugar(s), and/orlinkage(s) between the bases of a target cell-specific TRE. For example,certain point mutations within sequences of TREs have been shown todecrease transcription factor binding and stimulation of transcription.See Blackwood, et al. (1998) Science 281:60-63 and Smith et al. (1997)J. Biol. Chem. 272:27493-27496. One of skill in the art would recognizethat some alterations of bases in and around transcription factorbinding sites are more likely to negatively affect stimulation oftranscription and cell-specificity, while alterations in bases which arenot involved in transcription factor binding are not as likely to havesuch effects. Certain mutations are also capable of increasing TREactivity. Testing of the effects of altering bases may be performed invitro or in vivo by any method known in the art, such as mobility shiftassays, or transfecting vectors containing these alterations in TREfunctional and TRE non-functional cells. Additionally, one of skill inthe art would recognize that point mutations and deletions can be madeto a TRE sequence without altering the ability of the sequence toregulate transcription.

As used herein, a TRE derived from a specific gene is referred to by thegene from which it was derived and is a polynucleotide sequence whichregulates transcription of an operably linked polynucleotide sequence ina host cell that expresses said gene. For example, as used herein, a“human glandular kallikrein transcriptional regulatory element”, or“hKLK2-TRE” is a polynucleotide sequence, preferably a DNA sequence,which increases transcription of an operably linked polynucleotidesequence in a host cell that allows an hKLK2-TRE to function, such as acell (preferably a mammalian cell, even more preferably a human cell)that expresses androgen receptor, such as a prostate cell. An hKLK2-TREis thus responsive to the binding of androgen receptor and comprises atleast a portion of an hKLK2 promoter and/or an hKLK2 enhancer (i.e., theARE or androgen receptor binding site).

As used herein, a “probasin (PB) transcriptional regulatory element”, or“PB-TRE” is a polynucleotide sequence, preferably a DNA sequence, whichselectively increases transcription of an operably-linked polynucleotidesequence in a host cell that allows a PB-TRE to function, such as a cell(preferably a mammalian cell, more preferably a human cell, even morepreferably a prostate cell) that expresses androgen receptor. A PB-TREis thus responsive to the binding of androgen receptor and comprises atleast a portion of a PB promoter and/or a PB enhancer (i.e., the ARE orandrogen receptor binding site).

As used herein, a “prostate-specific antigen (PSA) transcriptionalregulatory element”, or “PSA-TRE”, or “PSE-TRE” is a polynucleotidesequence, preferably a DNA sequence, which selectively increasestranscription of an operably linked polynucleotide sequence in a hostcell that allows a PSA-TRE to function, such as a cell (preferably amammalian cell, more preferably a human cell, even more preferably aprostate cell) that expresses androgen receptor. A PSA-TRE is thusresponsive to the binding of androgen receptor and comprises at least aportion of a PSA promoter and/or a PSA enhancer (i.e., the ARE orandrogen receptor binding site).

As used herein, a “carcinoembryonic antigen (CEA) transcriptionalregulatory element”, or “CEA-TRE” is a polynucleotide sequence,preferably a DNA sequence, which selectively increases transcription ofan operably linked polynucleotide sequence in a host cell that allows aCEA-TRE to function, such as a cell (preferably a mammalian cell, evenmore preferably a human cell) that expresses CEA. The CEA-TRE isresponsive to transcription factors and/or co-factor(s) associated withCEA-producing cells and comprises at least a portion of the CEA promoterand/or enhancer.

As used herein, an “α-fetoprotein (AFP) transcriptional regulatoryelement”, or “AFP-TRE” is a polynucleotide sequence, preferably a DNAsequence, which selectively increases transcription (of an operablylinked polynucleotide sequence) in a host cell that allows an AFP-TRE tofunction, such as a cell (preferably a mammalian cell, even morepreferably a human cell) that expresses AFP. The AFP-TRE is responsiveto transcription factors and/or co-factor(s) associated withAFP-producing cells and comprises at least a portion of the AFP promoterand/or enhancer.

As used herein, an “a mucin gene (MUC) transcriptional regulatoryelement”, or “MUC1-TRE” is a polynucleotide sequence, preferably a DNAsequence, which selectively increases transcription (of anoperably-linked polynucleotide sequence) in a host cell that allows aMUC1-TRE to function, such as a cell (preferably a mammalian cell, evenmore preferably a human cell) that expresses MUC1. The MUC1-TRE isresponsive to transcription factors and/or co-factor(s) associated withMUC1-producing cells and comprises at least a portion of the MUC1promoter and/or enhancer.

As used herein, a “urothelial cell-specific transcriptional responseelement”, or “urothelial cell-specific TRE” is polynucleotide sequence,preferably a DNA sequence, which increases transcription of an operablylinked polynucleotide sequence in a host cell that allows aurothelial-specific TRE to function, i.e., a target cell. A variety ofurothelial cell-specific TREs are known, are responsive to cellularproteins (transcription factors and/or co-factor(s)) associated withurothelial cells, and comprise at least a portion of aurothelial-specific promoter and/or a urothelial-specific enhancer.Methods are described herein for measuring the activity of a urothelialcell-specific TRE and thus for determining whether a given cell allows aurothelial cell-specific TRE to function.

As used herein, a “melanocyte cell-specific transcriptional responseelement”, or “melanocyte cell-specific TRE” is polynucleotide sequence,preferably a DNA sequence, which increases transcription of an operablylinked polynucleotide sequence in a host cell that allows amelanocyte-specific TRE to function, i.e., a target cell. A variety ofmelanocyte cell-specific TREs are known, are responsive to cellularproteins (transcription factors and/or co-factor(s)) associated withmelanocyte cells, and comprise at least a portion of amelanocyte-specific promoter and/or a melanocyte-specific enhancer.Methods are described herein for measuring the activity of a melanocytecell-specific TRE and thus for determining whether a given cell allows amelanocyte cell-specific TRE to function.

As used herein, a target cell-specific TRE can comprise any number ofconfigurations, including, but not limited to, a target cell-specificpromoter; a target cell-specific enhancer; a target cell-specificpromoter and a target cell-specific enhancer; a target cell-specificpromoter and a heterologous enhancer; a heterologous promoter and atarget cell-specific enhancer; and multimers of the foregoing. Thepromoter and enhancer components of a target cell-specific TRE may be inany orientation and/or distance from the coding sequence of interest, aslong as the desired target cell-specific transcriptional activity isobtained. Transcriptional activation can be measured in a number of waysknown in the art (and described in more detail below), but is generallymeasured by detection and/or quantitation of mRNA or the protein productof the coding sequence under control of (i.e., operably linked to) thetarget cell-specific TRE.

As used herein, an “internal ribosome entry site” or “IRES” refers to anelement that promotes direct internal ribosome entry to the initiationcodon, such as ATG, of a cistron (a protein encoding region), therebyleading to the cap-independent translation of the gene. Jackson R J,Howell M T, Kaminski A (1990) Trends Biochem Sci 15(12):477-83) andJackson R J and Kaminski, A. (1995) RNA 1(10):985-1000). The presentinvention encompasses the use of any IRES element which is able topromote direct internal ribosome entry to the initiation codon of acistron. “Under translational control of an IRES” as used herein meansthat translation is associated with the IRES and proceeds in acap-independent manner. Examples of “IRES” known in the art include, butare not limited to IRES obtainable from picornavirus (Jackson et al.,1990, Trends Biochem Sci 15(12):477-483); and IRES obtainable from viralor cellular mRNA sources, such as for example, immunogloublinheavy-chain binding protein (BiP), the vascular endothelial growthfactor (VEGF) (Huez et al. (1998) Mol. Cell. Biol. 18(11):6178-6190),the fibroblast growth factor 2, and insulin-like growth factor, thetranslational initiation factor eIF4G, yeast transcription factors TFIIDand HAP4. IRES have also been reported in different viruses such ascardiovirus, rhinovirus, aphthovirus, HCV, Friend murine leukemia virus(FrMLV) and Moloney murine leukemia virus (MoMLV). As used herein,“IRES” encompasses functional variations of IRES sequences as long asthe variation is able to promote direct internal ribosome entry to theinitiation codon of a cistron. In preferred embodiments, the IRES ismammalian. In other embodiments, the IRES is viral or protozoan. In oneillustrative embodiment disclosed herein, the IRES is obtainable fromencephelomycarditis virus (ECMV) (commercially available from Novogen,Duke et al. (1992) J. Virol 66(3):1602-1609). In another illustrativeembodiment disclosed herein, the IRES is from VEGF. Table I and Table IIdisclose a variety of IRES sequences useful in the present invention. Insome embodiments, an adenovirus vector comprising an IRES exhibitsgreater specificity for the target cell than an adenovirus vectorcomprising a target cell-specific TRE operably linked to a gene andlacking an IRES. In some embodiments, specificity is conferred bypreferential transcription and/or translation of the first and secondgenes due to the presence of a target cell specific TRE. In otherembodiments, specificity is conferred by preferential replication of theadenovirus vectors in target cells due to the target cell-specific TREdriving transcription of a gene essential for replication.

A “multicistronic transcript” refers to an mRNA molecule which containsmore than one protein coding region, or cistron. A mRNA comprising twocoding regions is denoted a “bicistronic transcript.” The “5′-proximal”coding region or cistron is the coding region whose translationinitiation codon (usually AUG) is closest to the 5′-end of amulticistronic mRNA molecule. A “5′-distal” coding region or cistron isone whose translation initiation codon (usually AUG) is not the closestinitiation codon to the 5′ end of the mRNA. The terms “5′-distal” and“downstream” are used synonymously to refer to coding regions that arenot adjacent to the 5′ end of a mRNA molecule.

As used herein, “co-transcribed” means that two (or more) coding regionsof polynucleotides are under transcriptional control of singletranscriptional control element.

A “gene” refers to a coding region of a polynucleotide. A “gene” may ormay not include non-coding sequences and/or regulatory elements.

“Replicating preferentially”, as used herein, means that the adenovirusreplicates more in a target cell than a non-target cell. Preferably, theadenovirus replicates at a significantly higher rate in target cellsthan non target cells; preferably, at least about 2-fold higher,preferably, at least about 5-fold higher, more preferably, at leastabout 10-fold higher, still more preferably at least about 50-foldhigher, even more preferably at least about 100-fold higher, still morepreferably at least about 400- to 500-fold higher, still more preferablyat least about 1000-fold higher, most preferably at least about 1×10higher. Most preferably, the adenovirus replicates solely in the targetcells (that is, does not replicate or replicates at a very low levels innon-target cells).

As used herein, the term “vector” refers to a polynucleotide constructdesigned for transduction/transfection of one or more cell types.Vectors may be, for example, “cloning vectors” which are designed forisolation, propagation and replication of inserted nucleotides,“expression vectors” which are designed for expression of a nucleotidesequence in a host cell, or a “viral vector” which is designed to resultin the production of a recombinant virus or virus-like particle, or“shuttle vectors”, which comprise the attributes of more than one typeof vector.

The terms “polynucleotide” and “nucleic acid”, used interchangeablyherein, refer to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxyribonucleotides. These terms include a single-,double- or triple-stranded DNA, genomic DNA, cDNA, RNA, DNA-RNA hybrid,or a polymer comprising purine and pyrimidine bases, or other natural,chemically, biochemically modified, non-natural or derivatizednucleotide bases. The backbone of the polynucleotide can comprise sugarsand phosphate groups (as may typically be found in RNA or DNA), ormodified or substituted sugar or phosphate groups. Alternatively, thebackbone of the polynucleotide can comprise a polymer of syntheticsubunits such as phosphoramidates and thus can be a oligodeoxynucleosidephosphoramidate (P—NH2) or a mixed phosphoramidate-phosphodiesteroligomer. Peyrottes et al. (1996) Nucleic Acids Res. 24: 1841-8;Chaturvedi et al. (1996) Nucleic Acids Res. 24: 2318-23; Schultz et al.(1996) Nucleic Acids Res. 24: 2966-73. A phosphorothioate linkage can beused in place of a phosphodiester linkage. Braun et al. (1988) J.Immunol. 141: 2084-9; Latimer et al. (1995) Molec. Immunol. 32:1057-1064. In addition, a double-stranded polynucleotide can be obtainedfrom the single stranded polynucleotide product of chemical synthesiseither by synthesizing the complementary strand and annealing thestrands under appropriate conditions, or by synthesizing thecomplementary strand de novo using a DNA polymerase with an appropriateprimer. Reference to a polynucleotide sequence (such as referring to aSEQ ID NO) also includes the complement sequence.

The following are non-limiting examples of polynucleotides: a gene orgene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs,uracyl, other sugars and linking groups such as fluororibose andthioate, and nucleotide branches. The sequence of nucleotides may beinterrupted by non-nucleotide components. A polynucleotide may befurther modified after polymerization, such as by conjugation with alabeling component. Other types of modifications included in thisdefinition are caps, substitution of one or more of the naturallyoccurring nucleotides with an analog, and introduction of means forattaching the polynucleotide to proteins, metal ions, labelingcomponents, other polynucleotides, or a solid support. Preferably, thepolynucleotide is DNA. As used herein, “DNA” includes not only bases A,T, C, and G, but also includes any of their analogs or modified forms ofthese bases, such as methylated nucleotides, internucleotidemodifications such as uncharged linkages and thioates, use of sugaranalogs, and modified and/or alternative backbone structures, such aspolyamides.

A polynucleotide or polynucleotide region has a certain percentage (forexample, 80%, 85%, 90%, or 95%) of “sequence identity” to anothersequence means that, when aligned, that percentage of bases are the samein comparing the two sequences. This alignment and the percent homologyor sequence identity can be determined using software programs known inthe art, for example those described in Current Protocols in MolecularBiology (F. M. Ausubel et al., eds., 1987) Supplement 30, section7.7.18. A preferred alignment program is ALIGN Plus (Scientific andEducational Software, Pennsylvania), preferably using defaultparameters, which are as follows: mismatch=2; open gap=0; extend gap=2.

“Under transcriptional control” is a term well understood in the art andindicates that transcription of a polynucleotide sequence, usually a DNAsequence, depends on its being operably (operatively) linked to anelement which contributes to the initiation of, or promotes,transcription. “Operably linked” refers to a juxtaposition wherein theelements are in an arrangement allowing them to function.

An “E3 region” (used interchangeably with “E3”) is a term wellunderstood in the art and means the region of the adenoviral genome thatencodes the E3 products (discussed herein). Generally, the E3 region islocated between about 28583 and 30470 of the adenoviral genome. The E3region has been described in various publications, including, forexample, Wold et al. (1995) Curr. Topics Microbiol Immunol. 199:237-274.

A “portion” of the E3 region means less than the entire E3 region, andas such includes polynucleotide deletions as well as polynucleotidesencoding one or more polypeptide products of the E3 region. As usedherein, “cytotoxicity” is a term well understood in the art and refersto a state in which a cell's usual biochemical or biological activitiesare compromised (i.e., inhibited). These activities include, but are notlimited to, metabolism; cellular replication; DNA replication;transcription; translation; uptake of molecules. “Cytotoxicity” includescell death and/or cytolysis. Assays are known in the art which indicatecytotoxicity, such as dye exclusion, ³H-thymidine uptake, and plaqueassays.

An “E1B 19-kDa region” (used interchangeably with “E1B 19-kDa genomicregion”) refers to the genomic region of the adenovirus E1B geneencoding the E1B 19-kDa product. According to wild-type Ad5, the E1B19-kDa region is a 261 bp region located between nucleotide 1714 andnucleotide 2244. The E1B 19-kDa region has been described in, forexample, Rao et al., Proc. Natl. Acad. Sci. USA, 89:7742-7746. Thepresent invention encompasses deletion of part or all of the E1B 19-kDaregion as well as embodiments wherein the E1B 19-kDa region is mutated,as long as the deletion or mutation lessens or eliminates the inhibitionof apoptosis associated with E1B-19 kDa.

The term “selective cytotoxicity”, as used herein, refers to thecytotoxicity conferred by an adenovirus vector of the present inventionon a cell which allows or induces a target cell-specific TRE to function(a target cell) when compared to the cytotoxicity conferred by anadenoviral vector of the present invention on a cell which does notallow a target cell-specific TRE to function (a non-target cell). Suchcytotoxicity may be measured, for example, by plaque assays, byreduction or stabilization in size of a tumor comprising target cells,or the reduction or stabilization of serum levels of a markercharacteristic of the tumor cells, or a tissue-specific marker, e.g., acancer marker.

In the context of adenovirus, a “heterologous polynucleotide” or“heterologous gene” or “transgene” is any polynucleotide or gene that isnot present in wild-type adenovirus. Preferably, the transgene will alsonot be expressed or present in the target cell prior to introduction bythe adenovirus vector. Examples of preferred transgenes are providedbelow.

In the context of adenovirus, a “heterologous” promoter or enhancer isone which is not associated with or derived from an adenovirus gene.

In the context of adenovirus, an “endogenous” promoter, enhancer, or TREis native to or derived from adenovirus. In the context of promoter, an“inactivation” means that there is a mutation of or deletion in part orall of the of the endogenous promoter, ie, a modification or alterationof the endogenous promoter, such as, for example, a point mutation orinsertion, which disables the function of the promoter.

In the context of a target cell-specific TRE, a “heterologous” promoteror enhancer is one which is derived from a gene other than the gene fromwhich a reference target cell-specific TRE is derived.

A “host cell” includes an individual cell or cell culture which can beor has been a recipient of an adenoviral vector(s) of this invention.Host cells include progeny of a single host cell, and the progeny maynot necessarily be completely identical (in morphology or in total DNAcomplement) to the original parent cell due to natural, accidental, ordeliberate mutation and/or change. A host cell includes cellstransfected or infected in vivo or in vitro with an adenoviral vector ofthis invention.

“Replication” and “propagation” are used interchangeably and refer tothe ability of an adenovirus vector of the invention to reproduce orproliferate. These terms are well understood in the art. For purposes ofthis invention, replication involves production of adenovirus proteinsand is generally directed to reproduction of adenovirus. Replication canbe measured using assays standard in the art and described herein, suchas a burst assay or plaque assay. “Replication” and “propagation”include any activity directly or indirectly involved in the process ofvirus manufacture, including, but not limited to, viral gene expression;production of viral proteins, nucleic acids or other components;packaging of viral components into complete viruses; and cell lysis.

An “ADP coding sequence” is a polynucleotide that encodes ADP or afunctional fragment thereof. In the context of ADP, a “functionalfragment” of ADP is one that exhibits cytotoxic activity, especiallycell lysis, with respect to adenoviral replication. Ways to measurecytotoxic activity are known in the art and are described herein.

A polynucleotide that “encodes” an ADP polypeptide is one that can betranscribed and/or translated to produce an ADP polypeptide or afragment thereof. The anti-sense strand of such a polynucleotide is alsosaid to encode the sequence.

An “ADP polypeptide” is a polypeptide containing at least a portion, orregion, of the amino acid sequence of an ADP and which displays afunction associated with ADP, particularly cytotoxicity, moreparticularly, cell lysis. As discussed herein, these functions can bemeasured using techniques known in the art. It is understood thatcertain sequence variations may be used, due to, for example,conservative amino acid substitutions, which may provide ADPpolypeptides.

“Androgen receptor,” or AR, as used herein refers to a protein whosefunction is to specifically bind to androgen and, as a consequence ofthe specific binding, recognize and bind to an androgen response element(ARE), following which the AR is capable of regulating transcriptionalactivity. The AR is a nuclear receptor that, when activated, binds tocellular androgen-responsive element(s). In normal cells the AR isactivated by androgen, but in non-normal cells (including malignantcells) the AR may be activated by non-androgenic agents, includinghormones other than androgens. Encompassed in the term “androgenreceptor” are mutant forms of an androgen receptor, such as thosecharacterized by amino acid additions, insertions, truncations anddeletions, as long as the function is sufficiently preserved. Mutantsinclude androgen receptors with amino acid additions, insertions,truncations and deletions, as long as the function is sufficientlypreserved. In this context, a functional androgen receptor is one thatbinds both androgen and, upon androgen binding, an ARE.

A polynucleotide sequence that is “depicted in” a SEQ ID NO means thatthe sequence is present as an identical contiguous sequence in the SEQID NO. The term encompasses portions, or regions of the SEQ ID NO aswell as the entire sequence contained within the SEQ ID NO.

A “biological sample” encompasses a variety of sample types obtainedfrom an individual and can be used in a diagnostic or monitoring assay.The definition encompasses blood and other liquid samples of biologicalorigin, solid tissue samples such as a biopsy specimen or tissuecultures or cells derived therefrom, and the progeny thereof. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such as proteinsor polynucleotides. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, serum, plasma, biological fluid, and tissue samples.

An “individual” is a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, farm animals, sportanimals, rodents, primates, and pets.

An “effective amount” is an amount sufficient to effect beneficial ordesired results, including clinical results. An effective amount can beadministered in one or more administrations. For purposes of thisinvention, an effective amount of an adenoviral vector is an amount thatis sufficient to palliate, ameliorate, stabilize, reverse, slow or delaythe progression of the disease state.

A given TRE is “derived from” a given gene if it is associated with thatgene in nature.

“Expression” includes transcription and/or translation.

As used herein, the term “comprising” and its cognates are used in theirinclusive sense; that is, equivalent to the term “including” and itscorresponding cognates.

“A,” “an” and “the” include plural references unless the context clearlydictates otherwise.

Combination Adenoviral and Chemotherapeutic Therapy

Embodiments of the present invention include methods for theadministration of combinations of a target cell-specific adenoviralvector and at least one antineoplastic agent(s) to an individual withneoplasia. The antineoplastic agent includes those listed in Table 1.These include agents from each of the major classes ofchemotherapeutics, including but not limited to: alkylating agents,alkaloids, antimetabolites, anti-tumor antibiotics, nitrosoureas,hormonal agonists/antagonists and analogs, immunomodulators,photosensitizers, enzymes and others. In some embodiments, theantineoplastic is an alkaloid, an antimetabolite, an antibiotic or analkylating agent. In certain embodiments the antineoplastic agentsinclude, for example, thiotepa, interferon alpha-2a, and the M-VACcombination (methotrexate-vinblastine, doxorubicin, cyclophosphamide).Preferred antineoplastic agents include, for example, 5-fluorouracil,cisplatin, 5-azacytidine, and gemcitabine. Particularly preferredembodiments include, but are not limited to, doxorubicin, estramustine,etoposide, mitoxantrone, docetaxel (TAXOTERE™), paclitaxel (TAXOL™), andmitomycin C. TABLE 1 Antineoplastic Agents ALKYLATING ANTIBIOTICS ANTI-ALKALOIDS AGENTS AND ANALOGS METABOLITES ENZYMES IMMUNOMODULATORSDocetaxel Alkyl Sulfonates Aclacinomycins Folic Acid L-AsparaginaseInterferon-α (TAXOTERE ™) Analogs Etoposide Busulfan Actinomycin F₁Denopterin Pegasargase Interferon-β Irinotecan Improsulfan AnthramycinEdatrexate Interferon-γ Paclitaxel Piposulfan Azaserine MethotrexateInterferon-α-2a (TAXOL ™) Teniposide Bleomycins Piritrexim Interleukin-2Topotecan Aziridines Cactinomycin Pteropterin Lentinan VinblastineBenzodepa Carubicin Tomudex ® Propagermanium Vincristine CarboquoneCarzinophilin Trimetrexate PSK Vendesine Meturedepa ChromomycinsRoquinimex Vinorelbine Uredepa Dactinomycin Purine Analogs RituximabDaunorubicin Cladribine Sizofiran Ethylenimines and 6-Diazo-5-oxo-L-Fludarabine Trastuzumab Methylmelamines norleucine AltretamineDoxorubicin 6-Mercaptopurine Ubenimex Triethylenemelamine EpirubicinThiamiprine Triethylenephosphoramide Idarubicin ThioguanineTriethylenethiophosphoramide Menogaril Mitomycins MitoxantronePyrimidine Analogs Nitrogen Mycophenolic Acid Ancitabine MustardsChlorambucil Nogalamycin 5-Azacytidine Chlomaphazine Olivomycins6-Azauridine Cyclophosphamide Peplomycin Carmofur EstramustinePirarubicin Cytarabine Ifosfamide Plicamycin, DoxifluridineMechlorethamine Porfiromycin Emitefur Mechlorethamine PuromycinEnocitabine Oxide Hydrochloride Melphalan Streptonigrin FloxuridineNovembichin Streptozocin Fluorouracil Valrubicin Perfosfamide TubercidinGemcitabine Phenesterine Zinostatin Tegafur Prednimustine ZorubicinTrofosfamide Uracil Mustard Carboplatin Cisplatin Miboplatin OxaliplatinOthers Dacarbazine Mannomustine Mitobronitol Mitolactol ThiotepaPipobroman Temozolomide HORMONE ANTAGONISTS/AGONISTS & NITROSOUREASOTHERS ANALOGS PHOTOSENSITIZER Carmustine Aceglatone DexamethasonePorfimer Sodium Chlorozotocin Amsacrine Prednisone FotemustineBisantrene Lomustine Defosfamide Androgens Nimustine DemecolcineCalusterone Ranimustine Diaziquone Dromostanolone EflornithineEpitiostanol Elliptinium Mepitiostane Acetate Etoglucid TestolactoneFenretinide Finasteride Antiadrenals Gallium Nitrate AminoglutethimideHydroxyurea Mitotane Lonidamine Trilostane Miltefosine MitoguazoneAntiandrogens Mopidamol Bicalutamide Nitracrine Flutamide PentostatinNilutamide Phenamet Podophyllinic Antiestrogens Acid 2- EthylhydrazideProcarbazine Droloxifene Razoxane Tamoxifen Sobuzoxane ToremifeneSpirogermanium Exemestane Amsacrine Aromatase Inhibitors TretinoinAminoglutethimide Tenuazonic Anastrozole Acid Triaziquone Fadrozole2,2′,2″- Formestane Triclorotriethyl amine, Urethan Letrozole TopotecanEstrogens Fosfestrol Hexestrol Polyestradiol Phosphate LHRH AnalogsBuserelin Goserelin Leuprolide Triptorelin, Progestogens ChlormadinoneAcetate Medroxyprogesterone Megestrol Acetate Melengestrol

This section provides exemplary non-inclusive vector andchemotherapeutic combinations. The adenoviral vector used in the methodsdescribed herein is generally a replication-competent, target-cellspecific adenoviral vector-comprising an adenovirus gene essential forreplication under transcriptional control of a TRE, embodiments of whichare described infra. In some embodiments, the gene essential forreplication in the adenoviral vector is an early gene, preferably E1Aand/or E1B. In some embodiments the E1A and E1B genes are undertranscriptional of identical TREs. In other embodiments E1A and E1Bgenes are under transcriptional control of non-identical (orheterologous) TREs. In some embodiments, the adenovirus vector comprisesa transgene. In other embodiments, the adenovirus vector comprises ADP.In some embodiments, the adenovirus vector contains an E3 region.

In other embodiments, the adenovirus vectors comprise co-transcribedfirst and second genes under transcriptional control of a heterologous,target cell-specific transcriptional regulatory element (TRE), whereinthe second gene is under translational control of an internal ribosomeentry site (IRES).

The choice of adenoviral vector is primarily determined by the identityof the target cells and therefore the type of cancer to be treated. Asexplained below in detail, an adenoviral vector comprising a PSA-TRE,PB-TRE, or hKLK2-TRE would preferentially replicate in prostate cells;an adenoviral vector comprising a CEA-TRE would preferentially replicatein colorectal, gastric, pancreatic, breast and lung cells; an AFP-TREwould preferentially replicate in hepatoma cells, or liver tumors; aurothelial cell-specific TRE (such as uroplakin) would preferentiallyreplicate in bladder cells; a MUC-TRE would preferentially replicate inbreast cells; a melanocyte specific TRE (such as tyrosinase) wouldpreferentially replicate in melanoma cells.

Certain combinations of adenoviral vector and chemotherapeutic areparticularly effective for the treatment of particular types of cancerusing the methods described above. Based on our in vitro studies, notall combinations of target cell-specific adenoviral vector andchemotherapeutic result in synergy. As shown in Tables 5 and 6 inExamples 1 and 2, gemcitabine used with CV790 (a liver-specific viruswith E1A and E1B under transcriptional control of two identicalAFP-TREs) results in synergy. However, when gemcitabine is used withCV787 (a prostate-specific virus with E1A under transcriptional controlof a PB-TRE and E1B under transcriptional control of a PSE-TRE), synergyis not observed. 5-fluorouracil used with prostate-specific adenovirusCV787 results in synergy, but when used with liver-specific adenovirusCV790, synergy is not observed. In another embodiment disclosed herein,CV884 used with doxorubicin provides synergistic effect.

For example, with respect to treatment of prostate tumors, areplication-competent adenovirus in which a gene essential forreplication, preferably one or more early genes, is undertranscriptional control of a prostate specific TRE, as discussed below,may be used in conjunction with an antineoplastic agent that is in thealkaloid, antimetabolite, antibiotic, or alkylating agent class ofantineoplastics. Preferred examples of antineoplastic agents includedoxorubicin, mitoxantrone, paclitaxel, estramustine, etoposide anddocetaxel. Additional examples of antineoplastic agents include,5-fluorouracil or cisplatin.

In some embodiments of the adenovirus vector, E1A is undertranscriptional control of a prostate specific TRE. In other embodimentsE1B is under transcriptional control of a prostate specific TRE. In yetother embodiments, both E1A and E1B are under transcriptional control ofprostate specific TREs, which may or may not be the same sequence. Anexample of a suitable prostate specific replication-competent adenoviralvector is one comprising probasin (PB)-TRE controlling transcription ofE1A, and PSE-TRE controlling transcription of E1B, such as CV787 asdescribed in the examples. Particularly preferred embodiments includeadministration of the combination of 5-fluorouracil with a prostatespecific adenoviral vector in which a PSA-TRE controls transcription ofE1A. An example of a suitable adenoviral vector is CV706.

In some embodiments, a prostate specific adenoviral vector comprisingE1A and E1B under transcriptional control of two non-identical prostatespecific TREs, is administered in conjunction with any of the followingantineoplastic agents: paclitaxel; docetaxel; cisplatin; doxorubicin;estramustine; etoposide; mitoxantrone; and 5-fluorouracil. In someembodiments, the prostate specific TRE controlling transcription of E1Aand the prostate specific TRE controlling transcription of E1B areheterologous (i.e., of different sequence) with respect to each other.In some embodiments, the prostate specific TRE controlling transcriptionof E1A is derived from probasin (PB) and the prostate specific TREcontrolling transcription of E1B is derived from prostate specificantigen (PSA). In other embodiments, the prostate specific TREcontrolling transcription of E1A is derived from PSA, and the prostatespecific TRE controlling transcription of E1B is derived from probasin.PSA-derived and PB-derived TREs are described herein. In someembodiments, the adenoviral vector is CV787. In some embodiments, anIRES is translationally linked to an adenovirus gene essential forreplication, such as E1B and in preferred embodiments, E1B has itsendogenous promoter deleted and the IRES and E1B are in frame. In otherembodiments, the 19-kDa region of E1B is deleted.

Preferably, the prostate specific adenovirus vectors used in thesemethods also contains an E3 region, as described herein. For example,CV787 contains an E3 region.

With respect to liver tumors (hepatoma), any liver cell specificadenoviral vector may be used with the chemotherapeutic agents describedherein. Preferably, the TRE is derived from AFP. The liver specificadenovirus vectors may be used with chemotherapeutic agents from any ofthe following classes: antimetabolites (especially DNA damaging agents);alkylating agents (especially platinum containing agents); antibiotics;alkaloids. Preferably, the chemotherapeutic agent is an antibiotic suchas doxorubicin, mitoxantrone, or mitomycin-C. In some embodiments, thechemotherapeutic agent is paclitaxel, 5-azacytidine, gemcitabine,etoposide, or cisplatin. In some embodiments, E1A is undertranscriptional control of an AFP-TRE. In other embodiments, E1B isunder transcriptional control of an AFP-TRE. In yet other embodiments,E1A and E1B are under transcriptional control of two AFP-TREs (which maybe identical or non-identical). These vectors may or may not contain anE3 region. In some embodiments, E1A and E1B are co-transcribed and undertranscriptional control of an AFP-TRE, and E1B is under translationalcontrol of an IRES (with E1B promoter preferably deleted and preferablywith the IRES and E1B in frame). In other embodiments, the 19-kDa regionof E1B is deleted.

An example of a suitable vector is CV790, in which E1A and E1B are eachunder transcriptional control of identical AFP-TREs, and which furthercomprises an E3 region. Another example of a suitable vector is CV890,in which E1A and E1B are co-transcribed and under transcriptionalcontrol of an AFP-TRE wherein E1B is under translational control of anIRES. Vectors such as these have displayed in vivo synergy inconjunction with doxorubicin. Accordingly, in some embodiments, thetarget cell-specific adenoviral vector has E1A under transcriptionalcontrol of an AFP-TRE and E1B under translational control of an IRES,and further comprising an E3 region (such as CV890), and theantineoplastic is chosen from the antibiotic class of agents.Preferably, the antineoplastic is doxorubicin.

With respect to bladder tumors, any bladder cell specific adenoviralvector may be used with the chemotherapeutic agents described herein.Preferably, the TRE is derived from uroplakin. The bladder specificadenovirus vectors may be used with chemotherapeutic agents from any ofthe following classes: antimetabolites (especially DNA damaging agents);alkylating agents (especially platinum containing agents); antibiotics;alkaloids, hormone antagonists/agonists and analogs andimmunomodulators. Preferably, the chemotherapeutic agent is anantibiotic such as doxorubicin, mitoxantrone, bleomycin, valrubicin, ormitomycin C. In some embodiments, the chemotherapeutic agent ispaclitaxel, etoposide, docetaxel, gemcitabine, 5-fluorouracil,vinblastine, ifosfamide, thiotepa, interferon alpha-2a, methotrexate,goserelin, leuprolide, gallium nitrate, cyclophosphamide, vincristine,carboplatin or cisplatin. Preferably the chemotherapeutic agent iscisplatin, thiotepa, mitomycin C, or interferon alpha-2a. In someembodiments, E1A is under transcriptional control of an uroplakin-TRE.In other embodiments, E1B is under transcriptional control ofuroplakin-TRE. In yet other embodiments, E1A and E1B are undertranscriptional control of uroplakin-TREs (which may be identical ornon-identical). Examples of suitable vectors include CV829 and CV877, inwhich E1A and E1B are each under transcriptional control of heterologousuroplakin-derived TREs, and which further comprise an E3 region. Thesevectors may or may not contain an E3 region. In some embodiments of thevector, E1A and E1B are co-transcribed and under transcriptional controlof an uroplakin-TRE, and E1B is under translational control of an IRES(with the E1B promoter preferably deleted and preferably IRES and E1Bare in frame). In other embodiments, the 19-kDa region of E1B isdeleted. These vectors may or may not contain an E3 region. Examples ofvectors include CV874, CV875 and CV876, which comprise an E3 region.Another example includes CV884.

With respect to colorectal or breast tumors, any colorectal or breastcell specific adenoviral vector may be used with the chemotherapeuticagents described herein. Preferably, the TRE is derived from CEA. Thecolorectal or breast specific adenovirus vectors may be used withchemotherapeutic agents from any of the following classes:antimetabolites (especially DNA damaging agents); alkylating agents(especially platinum containing); antibiotics; alkaloids; hormoneantagonists/agonists and analogs (especially anti-estrogens).Preferably, the chemotherapeutic agent is an antibiotic such asdoxorubicin, mitoxantrone, epirubicin, or mitomycin-C. In someembodiments, the chemotherapeutic agent is paclitaxel, 5-fluorouracil,thiotepa, goserelin, exemestane, methotrexate, irinotecan, edatrexate,letrozole, leuprolide, cyclophosphamide, vinblastine, prednisone,docetaxel, paclitaxel, or cisplatin. Preferably the chemotherapeuticagent is a hormone or hormone analog anti-estrogen such as tamoxifen,anastrozole, exemestane or letrozole. In some embodiments, E1A is undertranscriptional control of an CEA-TRE. In other embodiments, E1B isunder transcriptional control of an CEA-TRE. In yet other embodiments,E1A and E1B are each under transcriptional control of CEA-TREs (whichmay be identical or non-identical). These vectors may or may not containan E3 region. In some embodiments, E1A is co-transcribed with E1B andunder transcriptional control of an CEA-TRE, and E1B is undertranslational control of an IRES (with the E1B promoter preferablydeleted and preferably IRES and E1B are in frame). In other embodiments,the 19-kDa region of E1B is deleted. These vectors may or may notcontain an E3 region. An example of a suitable vector is CV873, in whichE1A is under transcriptional control of a CEA-TRE and E1B is undertranslational control of an IRES, and which further comprises an E3region.

With respect to melanoma, any melanoma specific adenoviral vector may beused with the chemotherapeutic agents described herein. Preferably, theTRE is derived from tyrosinase. The melanoma specific adenovirus vectorsmay be used with chemotherapeutic agents from any of the followingclasses: antimetabolites (especially DNA damaging agents); alkylatingagents (especially platinum containing agents); antibiotics; alkaloids,hormone antagonists/agonists and analogs, nitrosoureas. In someembodiments, the chemotherapeutic agent is 5-fluorouracil, gemcitabine,doxorubicin, miroxantrone, mitomycin, dacarbazine, carmustine,vinblastine, lomustine, tamoxifen, docetaxel, paclitaxel or cisplatin.In some embodiments, E1A is under transcriptional control of atyrosinase-TRE. In other embodiments, E1B is under transcriptionalcontrol of a tyrosinase-TRE. In yet other embodiments, E1A and E1B areeach under transcriptional control of a tyrosinase-TREs (which may beidentical or non-identical). These vectors may or may not contain an E3region. In some embodiments, E1A is co-transcribed with E1B and undertranscriptional control of a tyrosinase-TRE, and E1B is undertranslational control of an IRES (with the E1B promoter preferablydeleted and preferably IRES and E1B are in frame). In other embodiments,the 19-kDa region of E1B is deleted. These vectors may or may notcontain an E3 region. An example is CV859, having E1A co-transcribedwith E1B and under transcriptional control of a tyrosinase-TRE and E1Bunder translational control of an IRES and an intact E3 region.

The specific choice of both the target cell-specific adenoviral vectorand the chemotherapeutic agent(s) is dependent upon, inter alia, thecharacteristics of the disease to be treated. These characteristicsinclude, but are not limited to, the type of cancer, location of thetumor, identity of the target cell, stage of the disease and theindividual's response to previous treatments, if any. It is wellestablished that certain antineoplastic agents are more efficacious forcertain types of cancer than others, for instance the use of tamoxifenin the treatment of breast cancer, the use of mitoxantrone orestramustine to treat prostate tumors or the use of doxorubicin and5-fluorouracil to treat hepatoma.

In addition to the use of single antineoplastic agents in combinationwith a particular adenoviral vector, the invention also includes the useof more than one agent in conjunction with an adenoviral vector. Table 2lists non-limiting examples of common combinations of antineoplasticagents. These combinations of antineoplastics when used to treatneoplasia are often referred to as combination chemotherapy and areoften part of a combined modality treatment which may also includesurgery and/or radiation, depending on the characteristics of anindividual's cancer. It is contemplated that the combinedadenoviral/chemotherapy of the present invention can also be used aspart of a combined modality treatment program. Preferred combinations ofchemotherapeutic agents include, but are not limited to, doxorubicin andcisplatin; doxorubicin; and mitomycin C; doxorubicin and mitoxantrone;and doxorubicin and paclitaxel (TAXOL™). In some embodiments, thesecombinations are used with an adenovirus specific for AFP producingcells, such as liver cells. An example of a suitable vector is CV790.

In other embodiments, preferred combinations of chemotherapeutic agentsinclude, but are not limited to, mitoxantrone and estramustine;paclitaxel (TAXOL™) and estramustine; and docetaxel (TAXOTERE™) andestramustine. In some embodiments, these combinations are used with anadenovirus specific for prostate cells, such as adenoviruses containingPSA-TRE, hKLK-TRE or PB-TRE. Examples of such adenoviruses include CV787and CV706.

In other embodiments, preferred combinations of chemotherapeutic agentsinclude, but are not limited to M-VAC (methotrexate-vinblastine,doxorubicin, cyclophosphamide), CISCA (cyclophosphamide, doxorubicin,cisplatin), CMV (cisplatin, methotrexate, vinblastine), CAP(cyclophosphamide, doxorubicin, cisplatin), or MVMJ (methotrexate,vinblastine, mitoxantrone, carboplatin). In some embodiments, thesecombinations are used with an adenovirus specific for bladder cells,such as those containing a uroplakin TRE. Examples of such adenovirusesinclude vectors such as CV829, CV874, CV875, CV876, CV877, and CV884described herein.

In other embodiments, preferred combinations include DBPT (dacarbazine,cisplatin, carmustine, tamoxifen), VDD (vinblastine, dacarbazine,cisplatin). In some embodiments these combinations are used withadenovirus vectors specific for melanoma, such as those containing atyrosinase-TREs. An example of a suitable vector is CV859, describedherein.

In other embodiments preferred combinations include levamisole and5-fluorouracil or leucovorin and fluorouracil. In particular embodimentsthese combinations can be used with colorectal specific adenoviralvectors, such as those containing a CEA-TRE. An example of a vector isCV873, described herein.

In other embodiments preferred combinations include CAF(cyclophosphamide, doxorubicin, 5-fluorouracil), CMF (cyclophosphamide,methotrexate, 5-fluorouracil), CNF (cyclophosphamide, mitoxantrone,5-fluorouracil), FAC (5-fluorouracil, doxorubicin, cyclophosphamide), MF(methotrexate, 5-fluorouracil, leucovorin), MV (mitomycin C,vinblastine), CMFP (cyclophosphamide, methotrexate, 5-fluorouracil,prednisone), VATH (vinblastine, doxorubicin, thiotepa, fluoxymesterone).In particular embodiments these combinations can be used with breastspecific adenoviral vectors, such as those containing a CEA-TRE. Anexample of such a vector is CV873, described herein.

Listed below are selected acronyms for combination cancer chemotherapyregimens comprising substances in The Merck Index. TABLE 2 CancerCombination Chemotherapy Drug Regimens Acronym Drug regimens AAcytarabine + doxorubicin ABP doxorubicin + bleomycin + prednisone ABVDdoxorubicin + bleomycin + vinblastine + dacarbazine AC doxorubicin +cyclophosphamide ACVB doxorubicin + cyclophosphamide + vindesine +bleomycin ADIC doxorubicin + dacarbazine APO doxorubicin + prednisone +vincristine + 6-mercaptopurine + asparaginase + methotrexate AVdoxorubicin˜vincristine AVDP asparaginase + vincristine + daunorubicin +prednisone BACOP bleomycin + doxorubicin + cyclophosphamide +vincristine + prednisone BAPP bleomycin + doxorubicin +, cisplatin +prednisone B - CAVe bleomycin + lomustine + doxorubicin + vincristineBCD methotrexate + doxorubicin + cisplatin BCP carmustine +cyclophosphamide + prednisone BCVPP carmustine + cyclophosphamide +‘vinblastine + procarbazine + prednisone B - DOPA bleomycin +dacarbazine + vincristine + prednisone + doxorubicin BEP bleomycin +etoposide + cisplatin BMP bleomycin + methotrexate + cisplatin BOLDbleomycin + vincristine + lomustine + dacarbazine CA cyclophosphamide +doxorubicin CAF cyclophosphamide + doxorubicin + fluorouracil CAMFcyclophosphamide + doxorubicin + methotrexate + fluorouracil CAPcyclophosphamide + doxorubicin + cisplatin CAP-BOP cyclophosphamide +doxorubicin + procarbazine + bleomycin + vincristine + prednisone CAVcyclophosphamide + doxorubicin + vincristine CAVE cyclophosphamide +doxorubicin + vincristine + etoposide CAVEP cyclophosphamide +doxorubicin + vincristine + etoposide + cisplatin CBV cyclophosphamide +carmustine + etoposide CC carboplatin + cyclophosphamide CD cytarabine +duanorubicin CFP cyclophosphamide + fluorouracil + prednisone CFPMVcyclophosphamide + fluorouracil + prednisone + methotrexate +vincristine CFPT cyclophosphamide + fluorouracil + prednisone +tamoxifen CHAD cyclophosphamide + hexamethylmelamine + doxorubicin +cisplatin CHAMOCA cyclophosphamide + hydroxyurea + dactinomycin +methotrexate + vincristine + doxorubicin CHAP-5 cyclophosphamide +hexamethylmelamine + doxorubicin + cisplatin CHF cyclophosphamide +hexamethylmelamine + fluorouracil ChIVPP chlorambucil + vinblastine +procarbazine + prednisone CHO cyclophosphamide + doxorubicin +vincristine CHOP cyclophosphamide + doxorubicin + vincristine +prednisone CHOP-B cyclophosphamide + doxorubicin + vincristine +prednisone + bleomycin CMF cyclophosphamide + methotrexate +fluorouracil CMFP cyclophosphamide + methotrexate + fluorouracil +prednisone CMFVP cyclophosphamide + methotrexate + fluorouracil +vincristine + prednisone C-MOPP cyclophosphamide + mechlorethamine +vincristine + procarbazine + prednisone CMV cisplatin + methotrexate +vinblastine COAP cyclophosphamide + vincristine + cytarabine +prednisolone CODE cisplatin + vincristine + doxorubicin + etoposideCOMLA cyclophosphamide + vincristine + methotrexate + cytarabine COMPcyclophosphamide + vincristine + methotrexate + prednisone COPcyclophosphamide + vincristine + prednisone COP-BLAM cyclophosphamide +vincristine + prednisone + bleomycin + doxorubicin + procarbazine COPPcyclophosphamide + vincristine + prednisone + procarbazine CVFcyclophosphamide + vincristine + fluorouracil CVP cyclophosphamide +vincristine + prednisone CVPP bleomycin + lomustine + doxorubicin +vinblastine CYVADIC cyclophosphamide + vincristine + doxorubicin +dacarbazine DCT daunorubicin + cytarabine + thioguanine DICEPcyclophosphamide + etoposide + cisplatin DVP duanorubicin +vincristine + prednisone EAP etoposide + doxorubicin + cisplatin EFPetoposide + fluorouracil + cisplatin ELF etoposide + leucovorin +fluorouracil EMA-CO etoposide + methotrexate + dactinomycin +cyclophosphamide + vincristine ESHAP etoposide + methylprednisolone +cytarabine + cisplatin FA fluorouracil + doxorubicin FAC fluorouracil +doxorubicin + cyclophosphamide FAM fluorouracil + doxorubicin +mitomycin C FAMTX fluorouracil + doxorubicin + methotrexate FAPfluorouracil + doxorubicin + cisplatin FEB fluorouracil + epirubicin +carmustine FUVAC fluorouracil + vinblastine + doxorubicin +cyclophosphamide HAD hexamethylmelamine + doxorubicin + cisplatin H-CAPhexamethylmelamine + cyclophosphamide + doxorubicin + cisplatin Hexa-CAFhexamethylmelamine + cyclophosphamide + methotrexate + fluorouracil ICEifosfamide + carboplatin + etoposide IMVP - 16 ifosfamide +methotrexate + etoposide LOPP chlorambucil + vincristine +procarbazine + prednisone LSA₂-L₂ cyclophosphamide + vincristine +prednisone + daunorubicin + methotrexate + cytarabine + thioguanine +colaspase + hydroxyurea + carmustine M - 2 vincristine + carmustine +cyclophosphamide + melphalan + prednisone MAC methotrexate +dactinomycin + chlorambucil MACC methotrexate + doxorubicin +cyclophosphamide + lomustine MACOP-B methotrexate + doxorubicin +cyclophosphamide + vincristine + prednisone + bleomycin M-BACODmethotrexate + bleomycin + doxorubicin + cyclophosphamide +vincristine + dexamethasone MBD methotrexate + bleomycin + cisplatin MCmitoxantrone + cytarabine MCF mitoxantrone + cyclophosphamide +fluorouracil MeCP methyl-CCNU + cyclophosphamide + prednisone MINEmesna + ifosfamide + mitoxantrone + etoposide MIP mitomycin +ifosfamide + cisplatin MM mercaptopurine + methotrexate MMMmitoxantrone + methotrexate + mitomycin MOP mechlorethamine +vincristine + procarbazine MOPP mechlorethamine + vincristine +procarbazine + prednisone MP melphalan + prednisone M-VAC methotrexate +vinblastine + doxorubicin + cisplatin MV mitroxantrone + etoposide MVPmitomycin + vindesine + cisplatin MPPP mechlorethamine + vinblastine +procarbazine + prednisone PAC cisplatin + doxorubicin + cyclophosphamidePC cisplatin + cyclophosphamide PCV procarbazine + lomustine +vincristine PE cisplatin + etoposide PEB cisplatin + etoposide +bleomycin PF L - PAM and fluorouracil PMF cisplatin + mitomycin C +fluorouracil ProMACE prednisone + methotrexate + doxorubicin +cyclophosphamide + etoposide ProMACE-CytaBOM prednisone + methotrexate +doxorubicin + cyclophosphamide + etoposide + cytarabine + bleomycin +vincristine + methotrexate ProMACE-MOPP prednisone + methotrexate +doxorubicin + cyclophosphamide + etoposide + mechlorethamine +vincristine + procarbazine + prednisone PVP - 16B VP - 16 + bleomycin +cisplatin PVB cisplatin + vinblastine + bleomycin SMF streptozocin +mitomycin + fluorouracil TC thioguanine + cytarabine VAB-6 vinblastine +dactinomycin + bleomycin + cisplatin + cyclophosphamide VACvincristine + dactinomycin + cyclophosphamide VAD vincristine +doxorubicin + dexamethasone VAMP vincristine + prednisone +methotrexate + 6-mercaptopurine VAP-cyclo vincristine + doxorubicin +prednisolone + cyclophosphamide VBAP vincristine + carmustine +dexamethasone + prednisone VCAP vincristine + cyclophosphamide +doxorubicin + prednisone VIP vindesine + ifosfamide + cisplatin VMFetoposide + methotrexate + fluorouracil VP vindesine + cisplatinAdministration and Assessment

There are a variety of delivery methods for the administration ofantineoplastic agents, which are well known in the art, including oraland parenteral methods. There are a number of drawbacks to oraladministration for a large number of antineoplastic agents, includinglow bioavailability, irritation of the digestive tract and the necessityof remembering to administer complicated combinations of drugs. Themajority of parenteral administration of antineoplastic agents isintravenously, as intramuscular and subcutaneous injection often leadsto irritation or damage to the tissue. Regional variations of parenteralinjections include intra-arterial, intravesical, intra-tumor,intrathecal, intrapleural, intraperitoneal and intracavity injections.

Delivery methods for chemotherapeutic agents include intravenous,intraparenteral and introperitoneal methods as well as oraladministration. Intravenous methods also include delivery through a veinof the extremities as well as including more site specific delivery,such as an intravenous drip into the portal vein of the liver. Otherintraparenteral methods of delivery include direct injections of anantineoplastic solution, for example, subcutaneously, intracavity orintra-tumor.

Delivery of adenoviral vectors is discussed infra and is generallyaccomplished by either site-specific injection or intravenously.Site-specific injections of either vector or antineoplastic agent(s) mayinclude, for example, injections into the portal vein of the liver aswell as intraperitoneal, intrapleural, intrathecal, intra-arterial,intra-tumor injections or topical application. These methods are easilyaccommodated in treatments using the combination of adenoviral vectorsand chemotherapeutic agents.

The adenoviral vectors may be delivered to the target cell in a varietyof ways, including, but not limited to, liposomes, general transfectionmethods that are well known in the art (such as calcium phosphateprecipitation or electroporation), direct injection, and intravenousinfusion. The means of delivery will depend in large part on theparticular adenoviral vector (including its form) as well as the typeand location of the target cells (i.e., whether the cells are in vitroor in vivo).

If used as a packaged adenovirus, adenovirus vectors may be administeredin an appropriate physiologically acceptable carrier at a dose of about10⁴ to about 10¹⁴. The multiplicity of infection will generally be inthe range of about 0.001 to 100. If administered as a polynucleotideconstruct (i.e., not packaged as a virus) about 0.01 μg to about 1000 μgof an adenoviral vector can be administered. The adenoviral vector(s)may be administered one or more times, depending upon the intended useand the immune response potential of the host, and may also beadministered as multiple, simultaneous injections. If an immune responseis undesirable, the immune response may be diminished by employing avariety of immunosuppressants, so as to permit repetitiveadministration, without a strong immune response. If packaged as anotherviral form, such as HSV, an amount to be administered is based onstandard knowledge about that particular virus (which is readilyobtainable from, for example, published literature) and can bedetermined empirically.

Generally, the adenovirus and chemotherapeutic agent are administered ascompositions in a pharmaceutically acceptable excipient (and may or maynot be in the same compositions), including, but not limited to, salinesolutions, suitable buffers, preservatives, stabilizers, and may beadministered in conjunction with suitable agents such as antiemetics. Insome embodiments, an effective amount of an adenoviral vector and aneffective amount of at least one antineoplastic agent are combined witha suitable excipient and/or buffer solutions and administeredsimultaneously from the same solution by any of the methods listedherein or those known in the art. This may be applicable when theantineoplastic agent does not compromise the viability and/or activityof the adenoviral vector itself. Where more than one antineoplasticagent is administered, the agents may be administered together in thesame composition; sequentially in any order; or, alternatively,administered simultaneously in different compositions. If the agents areadministered sequentially, administration may further comprise a timedelay.

The chemotherapeutic agent and adenovirus may be administeredsimultaneously or sequentially, with various time intervals forsequential administration. In some embodiments, chemotherapeuticagent(s) and adenovirus vector(s) are administered simultaneously. Asshown in the Examples, at least some antineoplastics do not appear tocompromise viral replication or specificity. The method of delivery willdepend upon both the choice of the adenoviral vector andchemotherapeutic agent(s) and by the characteristics of the cancer undertreatment.

In other embodiments, a chemotherapeutic agent and adenoviral vector canbe administered sequentially. This may be appropriate, for example, ininstances where the antineoplastic agent is an alkylating agent,antimetabolite, nitrosourea or other DNA damaging agent which maycompromise the viability and/or activity or the viral vector, or ininstances in which it has been indicated that sequential administrationoptimizes effectiveness of the combination therapy. Sequentialadministration may be in any order, and accordingly encompasses theadministration of an effective amount of an adenoviral vector first,followed by the administration of an effective amount of thechemotherapeutic agent. The interval between administration ofadenovirus and chemotherapeutic agent may be in terms of at least (or,alternatively, less than) minutes, hours, or days. Sequentialadministration also encompasses administration of a chosenantineoplastic agent followed by the administration of the adenoviralvector. The interval between administration may be in terms of at least(or, alternatively, less than) minutes, hours, or days.

Administration of the above-described methods may also include repeatdoses or courses of target-cell specific adenovirus and chemotherapeuticagent depending, inter alia, upon the individual's response and thecharacteristics of the individual's disease. Repeat doses may beundertaken immediately following the first course of treatment (i.e.,within one day), or after an interval of days, weeks or months toachieve and/or maintain suppression of tumor growth. A particular courseof treatment according to the above-described methods, for example,combined adenoviral and chemotherapy, may later be followed by a courseof combined radiation and adenoviral therapy.

Generally, an effective amount of adenovirus vector and chemotherapeuticagent(s) is administered, i.e., amounts sufficient to achieve thedesired result, based on general empirical knowledge of a population'sresponse to such amounts. Some individuals are refractory to thesetreatments, and it is understood that the methods encompassadministration to these individuals. The amount to be given depends,inter alia, on the type of cancer, the condition of the individual, theextent of disease, the route of administration, how many doses will beadministered, and the desired objective.

A chemotherapeutic agent(s) is administered in a physiologicallyacceptable carrier appropriate to the method of delivery, as are knownin the art and described herein. The amount of chemotherapeutic agent(s)administered is determined by the characteristics of the individual'sdisease, the method of delivery and the weight, age, general health andresponse of the individual. In some embodiments the amount ofchemotherapeutic agent(s) administered will be the dosage known in theart to be effective given the characteristics of the individual andtheir disease. In other embodiments, due to the synergistic effect ofthe combination of adenoviral vector and chemotherapeutic agent, theamount of chemotherapeutic agent(s) administered will be about 2×, about5×, about 10×, or about 5× less than that known in the art to beeffective for the particular individual and characteristics of thedisease. In some embodiments, the amount of chemotherapeutic agent(s)administered will be about 20×, about 50×, about 100× or about 100× lessthan that known in the art to be effective for the particular individualand characteristics of the disease. Dosages include courses ofchemotherapy and repeat administrations of the chemotherapeutic agent(s)over the course of days, weeks or months and may include an increase ordecrease in the interval between doses during administration of thecourse of chemotherapy, or increases or decreases in the actual amountof chemotherapeutic agent administered.

Examples of dosages known in the art for chemotherapeutic agentsinclude, but are not limited to, doses of 60-75 mg/m² for doxorubicin at21 day intervals when administered as a single agent, and doxorubicindoses of 40-60 mg/m² when administered as a component in a combinationof chemotherapeutic agents. Typical doses known in the art for cisplatinare from 20 mg/m² to 100 mg/m²; for etoposide 35-100 mg/m²; forpaclitaxel 135-175 mg/m²; for docetaxel 60-100 mg/m²; for mitomycin C3040 mg/m²; gemcitabine 1000-1250 mg/m²; mitoxantrone 12-14 mg/m² percycle, 12-212 mg/m² cumulative over course of treatment; thiotepa0.3-0.8 mg/kg; 5-azacytidine 50-200 mg/m²/day; 5-fluorouracil 7-12mg/kg/day, not more than 800 mg/day. These dose may be administered on avariety of schedules known to those of skill in the art and depending onthe response of the individual and the characteristic of the individualcancer.

Any of the methods described herein may further be used in conjunctionwith combined modality treatment for suppressing tumor growth. Suchcombined modality treatment may or may not include surgery as acomponent of the treatment.

Assessment may be determined by any of the techniques known in the art,including diagnostic methods such as imaging techniques, analysis ofserum tumor markers (which may be measured, for example, by ELISA),biopsy (which could indicate the presence of killed tumor cells), andthe presence, absence or amelioration of tumor associated symptoms.

Compositions and Kits of Adenoviral Vectors and Chemotherapeutic Agents

The invention also includes compositions comprising at least oneantineoplastic agent, such as those listed in Table 1, and a targetcell-specific adenoviral vector(s) as described herein, where thestability, activity, and/or viability of the adenoviral vector is notcompromised by the antineoplastic agent(s) (ie, the adenovirus vectorretains some to all activity). These compositions can further comprisesuitable pharmaceutical such as, saline solutions, suitable buffers,preservatives, stabilizers.

In some embodiments, the composition comprises a target cell-specificadenoviral vector comprising E1A under transcriptional control of aPB-TRE, E1B under transcriptional control of a PSA-TRE, furthercomprising an E3 region (such as CV787) and the antineoplastic is5-fluorouracil or cisplatin. In other embodiments, the antineoplastic isdoxorubicin, estramustine, etoposide, mitoxantrone, docetaxel(TAXOTERE™) or paclitaxel (TAXOL™). In other embodiments, thecomposition comprises an adenovirus vector comprising E1A undertranscriptional control of a PSA-TRE (such as CV706) and the compositionfurther comprises 5-fluorouracil or cisplatin. In other embodiments, theantineoplastic is doxorubicin, estramustine, etoposide, mitoxantrone,docetaxel (TAXOTERE™) or paclitaxel (TAXOL™). In other embodiments, thecomposition comprises an adenoviral vector comprising an early geneunder transcriptional control of an AFP-TRE (for example, E1A undertranscriptional control of an AFP-TRE), E1B under transcriptionalcontrol of an AFP-TRE, an intact E3 region (such as CV790) and thecomposition further comprises 5-azacytidine, cisplatin, etoposide orgemcitabine, doxorubicin, mitomycin C, mitoxantrone, paclitaxel or acombination of antineoplastic agents such as, doxorubicin and cisplatin,or doxorubicin and mitomycin C or doxorubicin and mitoxantrone ordoxorubicin and paclitaxel (TAXOL™).

In some embodiments, the adenovirus vector comprises co-transcribedfirst and second genes, preferably adenovirus genes, undertranscriptional control of a heterologous, target cell-specifictranscriptional regulatory element (TRE), wherein the second gene isunder translational control of an internal ribosome entry site (IRES).

Kits comprising the combined antineoplastic agent(s), targetcell-specific adenoviral vector, and suitable excipient, packaging, andlabeling are also included in the present invention. The kit providessuitable dosages of each of the antineoplastic agent(s) and adenoviralvector. Embodiments include kits comprising, for example, all of thecompositions listed above. The kits preferably contain instructions foradministration to individuals for appropriate cancer to effectsuppression of tumor growth.

In some embodiments, the chemotherapeutic agent and adenoviral vectorare packaged separately in appropriate packaging. In other embodiments,the chemotherapeutic agent and adenoviral vector are packaged together.Examples of suitable agents and adenoviral vectors have been discussedabove and are described herein.

Combination Adenoviral and Radiation Therapy

The invention also provides combination methods which employ thereplication competent target cell specific adenoviral vectors asdescribed herein and radiation. As explained in more detail in Example6, the combined treatment of neoplasia with a target cell-specificadenoviral vector and radiation results in a synergistic effect, withearlier eradication of the tumor compared to no treatment, radiationalone or virus alone. When used in combination with target cell-specificadenoviral vectors, the type of radiation treatment used is dependentupon the characteristics of the individual cancer being treated. Thechoice of suitable radiation therapy is well known by a person skilledin the art and decided on an individual basis. The choice of the targetcell-specific adenoviral vector is largely governed by the identity ofthe target (neoplastic) cells and includes X-rays, gamma rays, alphaparticles, beta particles, radioactive isotopes, photons, neutrons,electrons and other forms of ionizing radiation. Sources of radiationinclude Americium, chromic phosphate, radioactive Cobalt,¹³¹I-ethiodized oil, Gold (radioactive, colloidal) iobenguane, Radium,Radon, sodium iodide (radioactive), sodium phosphate (radioactive), and¹³⁷Cesium. Radioimmunotherapy can also be used. In some embodiments,radiation therapy includes use of one or more radiosensitizing agent(s)or radiation protectants.

Accordingly, the present invention includes methods of suppressing tumorgrowth in an individual comprising the following steps:

-   -   a) administration of an effective amount of a        replication-competent target cell-specific adenoviral vector to        an individual with neoplasia; and    -   b) administration of an effective amount of an appropriate        course of radiation wherein radiation includes X-rays, gamma        rays, alpha particles, beta particles, electrons, photons,        neutrons, other ionizing radiation and radioactive isotopes.

In some embodiments, step (a) is performed before step (b). In otherembodiments, step (b) is performed before step (a). In otherembodiments, steps (a) and (b) are performed simultaneously.

The replication-competent target cell-specific adenoviral vector may beany of the replication-competent target cell-specific adenoviral vectorsdisclosed herein, comprising a gene essential for replication,preferably an early gene, under transcriptional control of a TRE.Preferably, the gene essential for replication is E1A or E1B or both.Discussion of exemplary embodiments of suitable adenoviral vectors inthe previous section, as well as the section describing adenovirusvectors below, are applicable to these methods.

In some embodiments, the gene essential for replication is E1A or E1Band in some embodiments, the vector comprises both E1A and E1B undertranscriptional control of a cell-specific TRE. In some embodiments, theE1A and E1B genes are under transcriptional control of the same orsimilar TREs. The vectors may or may not include an E3 region. In someembodiments, the adenovirus vector comprises co-transcribed first andsecond genes, preferably adenovirus genes, under transcriptional controlof a heterologous, target cell-specific transcriptional regulatoryelement (TRE), wherein the second gene is under translational control ofan internal ribosome entry site (IRES). In some embodiments, the firstand second genes are E1A and E1B, respectively. In this embodiment it ispreferred that E1B has its endogenous promoter deleted and in oneembodiment, IRES and E1B are in frame.

In other embodiments, the adenovirus vector comprises E1A wherein theE1A promoter is deleted and wherein the E1A gene is undertranscriptional control of a target cell-specific TRE. In otherembodiments, the adenovirus gene is E1B wherein the E1B promoter isdeleted and wherein the E1B gene is under transcriptional control of atarget cell-specific TRE. In other embodiments, the vector comprises E1Awherein the E1A promoter is deleted and E1B wherein the E1B promoter isdeleted.

In other embodiments, an enhancer element for the first and/or secondadenovirus genes is deleted. In some embodiments, the E1A enhancer isdeleted. In yet other embodiments, the E1A promoter is deleted and E1Aenhancer I is deleted. In further embodiments, the TRE has itsendogenous silencer element deleted. In other embodiments, theadenovirus vector comprises E1B having a deletion in the 19-kDa region.These embodiments apply to any and all methods described herein.

Administration and Assessment

As is well-known in the art, radiation therapy includes treatment withX-rays and gamma-rays, as well as alpha and beta particles, photons,electrons, neutrons, implants of radioactive isotopes and other forms ofionizing radiation. Recent experimental therapy employs monoclonalantibodies specific to the malignant tumor to deliver radioactiveisotopes directly to the site of the tumor, termed radioimmunotherapy.The most common type of radiation treatment is radiation directed to thebody area containing the neoplastic tumor, which is known as regional orlocal radiation therapy.

The combined modality treatment of radiation and target cell-specificadenoviral therapy can be carried out in a number of ways, includingdelivery of the adenoviral vector followed by radiation therapy, orwhere vector delivery is followed by a time delay of seconds, minutes,hours or days and before radiation treatment. The combined modalitytreatment also incorporates administration of the radiation treatmentfollowed by the adenoviral treatment, including but not necessarilyrequiring a time interval between radiation treatment and delivery ofthe adenovirus, of seconds, minutes, hours or days.

Repeat dosages of adenoviral vector and/or radiation may beadministered. Administration of adenovirus vectors has been describedabove. Administration of radiation therapy can include methods wellknown in the art, such as internal and external radiation therapy.External therapy includes the administration of radiation viahigh-energy external beam radiation, administered either regionally(locally) to the tumor site or whole body irradiation. Examples ofinternal radiation (brachytherapy) include the implantation ofradioactive isotopes in permanent, temporary, sealed, unsealed,intracavity or interstitial implants. The choice of implant isdetermined by the characteristics of the neoplasia, including thelocation and extent of the tumor. The choice between external orinternal radiation treatment and type of external radiation treatment isalso determined by the characteristics of the neoplasia and can bedetermined by those skilled in the art. An additional type of radiationtherapy is radioimmunotherapy in which radioisotopes are attached tomonoclonal antibodies specific for the tumor cells.

The amount/course of radiation administered to the individual isdetermined by the characteristics of the individual's disease, themethod of delivery and the weight, age, general health and response ofthe individual. For radiation therapy in particular, the location of thetumor is a determining factor in the administration of radiation, as theradiosensitivity of the tumor and surrounding tissue are variableaccording to tissue type (see Table 3), oxygen supply and other factors.In some embodiments the amount of radiation administered will be thedosage known in the art to be effective given the characteristics of theindividual and the disease. In other embodiments, the amount ofradiation administered will be about 2×, about 5×, about 10×, or about15× less than that known in the art to be effective for the particularindividual and characteristics of the disease. In some embodiments, theamount of radiation administered will be about 20×, about 50×, about100× or about 1000× less than that known in the art to be effective forthe particular individual and characteristics of the disease.

Radiation treatment may also entail the administration of aradiosensitizing agent or radioprotectant to facilitate the treatment.Recent evidence suggests that the antineoplastic agent TAXOL™(paclitaxel) may function as a radiosensitizer. Liebmann et al., J.National Cancer Inst. 86:441, 1994. Similar evidence has been found forTAXOTERE™ (docetaxel). Creane et al., Int. J. Radiat. Biol. 75:731,1999; Sikov et al., Front. Biosci. May 1: 221, 1997. Other radiationsensitizers include E2F-1, anti-ras single chain antibody, p53, GM-CSF,and cytosine deaminase. A tumor specific adenovirus may further comprisea radiation sensitizer, such as p53 for example, or a chemo sensitizer.

Repeat doses may be undertaken immediately following the first course oftreatment or after an interval of days, weeks or months to achievesuppression of tumor growth. A particular course of treatment accordingto the above-described methods, for example, combined adenoviral andradiation therapy, may later be followed by a course of combinedchemotherapy and adenoviral therapy. TABLE 3 Radiosensitivity of VariousTissues Relative Tumor or Tissue Type Radiosensitivity lymphoma,leukemia, seminoma, high dysgerminoma squamous cell, cancer of thefairly high oropharyngeal glottis, bladder, skin & cervical epitheliaadenocarinomas of the alimentary tract vascular & connective tissue(elements of medium all tumors) secondary neurovascularization,astrocytomas salivary gland tumors, hepatoma, renal fairly low cancer,pancreatic cancer, chondrosarcoma, osteogenic sarcomas rhabdomyosarcoma,leiomyosarcoma & low ganglioneurofibrosarcoma

Assessment may be determined by any of the techniques known in the art,including diagnostic methods such as imaging techniques, analysis ofserum tumor markers, biopsy, the presence, absence or amelioration oftumor associated symptoms.

Combination Treatment with Adenoviral, Chemotherapy and Radiation

Chemotherapy and radiation are commonly used as components of a combinedmodality treatment, and the choice of chemotherapeutic agent(s) and typeand course of radiation therapy is generally governed by thecharacteristics of the individual cancer and the response of theindividual. While target cell-specific adenoviral vectors can be usedwith either radiation or chemotherapy, as separate courses of treatment,they can also be combined with both methods of treatment in the samecourse of therapy. Accordingly, the present invention encompassescombinations of the methods discussed above.

Accordingly, the invention includes methods for suppressing tumor growthin an individual comprising the following steps, in any order:

-   -   a) administering to the individual an effective amount of a        target cell-specific adenoviral vector and at least one        antineoplastic agent; and    -   b) administering an effective amount of an appropriate course of        radiation therapy to the individual.

The method may further comprise the step of:

-   -   c) administering to the individual an additional dose of the        adenoviral/chemotherapeutic solution or radiation as necessary        to treat the individual's neoplasia.

The method may further comprise time delays after any one of steps a),b) and c). A time delay interval may be days, weeks or months.

The antineoplastic may be chosen from the agents listed in Table 1 or acombination of agents may be chosen from the list in Table 2. Additionalagents or combinations of agents known to those of skill in the art mayalso be used. The replication-competent target cell-specific adenoviralvector is chosen from the replication-competent target cell-specificadenoviral vectors disclosed herein.

In preferred embodiments the gene essential for replication in theadenoviral vector is an early gene. Even more preferably the geneessential for replication is E1A or E1B or both. In particularlypreferred embodiments the E1A and E1B genes are under transcriptional ofthe same or similar TREs. The vector may or may not contain an E3region.

In some embodiments, the adenovirus vector comprises co-transcribedfirst and second genes under transcriptional control of a heterologous,target cell-specific transcriptional regulatory element (TRE), whereinthe second gene is under translational control of an internal ribosomeentry site (IRES). An adenovirus vector may further comprise E3.

In particular embodiments of the above described methods, the adenoviralgene(s) essential for replication is under the control of TRE(s)specific for target cells such as, but not limited to liver, prostate,bladder, colorectal, breast or melanoma cells.

In certain preferred embodiments of the above described methods, theadenoviral gene(s) essential for replication is under the control of aTRE(s) such as, but not limited to the PB-TRE, PSA-TRE, the MUC-TRE, theAFP-TRE, the CEA-TRE, the hKLK2-TRE, tyrosinase-TRE, and uroplakin-TRE,as described herein.

Illustrative embodiments of target cell-specific adenoviral vectorsinclude CV787, CV790, CV890, CV706, CV829, CV859, CV873, CV874, CV875,CV876, CV877, and CV884 as described herein.

In a preferred embodiment, the adenoviral vector comprises a prostatespecific TRE or a liver specific TRE and at least one of thechemotherapeutic agents is from the alkaloid class.

In another preferred embodiment, the adenoviral vector comprises aprostate specific TRE or a liver specific TRE and at least one of thechemotherapeutic agents is paclitaxel (TAXOL™) or docetaxel (TAXOTERE™)or a paclitaxel derivative.

In another preferred embodiment the adenoviral vector comprises aurothelial specific TRE and least one of the chemotherapeutic agents ispaclitaxel (TAXOL™) or docetaxel (TAXOTERE™) or a paclitaxel derivative.

Administration and Assessment

Administration of adenoviral vectors, chemotherapeutic agents andradiation has been described above. The choice of the adenoviral vector,chemotherapeutic agent(s) and radiation are dependent on thecharacteristics of the individual cancer and the individual's responseto therapy. Such considerations are known to those skilled in the art.The invention encompasses embodiments which include thereplication-competent target cell-specific adenoviral vectors discussedherein as well as those known to persons of skill in the art. Theinvention also encompasses embodiments which include the combinations oftarget cell-specific adenoviral vectors and chemotherapeutic agentsdiscussed herein which can be further combined with radiation therapy.

The above-described methods include administration of the adenoviralvector, radiation and chemotherapeutic(s) in any order and may includesequential administration or simultaneous administration of all or someof the components (i.e. simultaneous administration of chemotherapy andadenovirus followed sequentially by radiation therapy or sequentialadministration of adenovirus first, radiation second and thirdly,chemotherapy, etc.).

Repeat doses may be undertaken immediately following the first course oftreatment or after an interval of days, weeks or months to achievesuppression of tumor growth. Repeat doses of a particular component ofthe therapy may also be administered before the administration of theremaining components (i.e. administration of multiple doses ofchemotherapeutic agent(s) followed by sequential administration ofradiation and adenovirus or administration of multiple doses ofradiation therapy followed by simultaneous administration ofchemotherapy and adenovirus, etc.). A particular course of treatmentaccording to the above-described methods, for example, combinedadenoviral, chemotherapeutic and radiation therapy, may later befollowed by a course of combined chemotherapy and adenoviral therapy.

Any of the methods described herein may further be used in conjunctionwith combined modality treatment for suppressing tumor growth. Suchcombined modality treatment may include surgery as a component of thetreatment.

Assessment of the suppression of tumor growth may be determined by anyof the techniques known in the art, including diagnostic methods such asimaging techniques, analysis of serum tumor markers, biopsy, thepresence, absence or amelioration of tumor associated symptoms.

Adenoviral Vectors

The adenoviral vectors used in the methods described herein arereplication-competent target-cell specific adenoviral vectors comprisingan adenovirus gene, preferably a gene essential for replication undertranscriptional control of a target cell specific TRE. The vector may ormay not include an E3 region. In other embodiments, an adenovirus vectoris a replication competent, target cell specific vector comprising E1B,wherein E1B has a deletion of part or all of the 19-kDa region.

In some embodiments the adenoviral gene essential for replication is anearly gene, preferably E1A or E1B or both.

In some embodiments, the adenovirus vector comprises co-transcribedfirst and second genes under transcriptional control of a heterologous,target cell-specific transcriptional regulatory element (TRE), whereinthe second gene is under translational control of an internal ribosomeentry site (IRES). The adenovirus vector may further comprise E3.

The adenovirus vectors used in this invention replicate preferentiallyin TRE functional cells referred to herein as target cells. Thisreplication preference is indicated by comparing the level ofreplication (i.e., titer) in cells in which the TRE is active to thelevel of replication in cells in which the TRE is not active (i.e., anon-target cell). The replication preference is even more significant,as the adenovirus vectors used in the invention actually replicate at asignificantly lower rate in TRE non-functional cells than wild typevirus. Comparison of the adenovirus titer of a target cell to the titerof a TRE inactive cell type provides a key indication that the overallreplication preference is enhanced due to the replication in targetcells as well as depressed replication in non-target cells. This isespecially useful in the cancer context, in which targeted cell killingis desirable. The TRE's preferably control genes necessary forreplication, where the gene(s) necessary for replication is an earlygene(s) of the adenovirus, preferentially the E1A or E1B genes.Particularly preferred embodiments include where TRE's control both theE1A and E1B genes within the same viral construct. In anotherparticularly preferred embodiment, the adenovirus vector comprisesco-transcribed first and second genes under transcriptional control of aheterologous, target cell-specific transcriptional regulatory element(TRE), wherein the second gene is under translational control of aninternal ribosome entry site (IRES). In this embodiment, it is preferredthat the second gene has its endogenous promoter mutated or deleted andin one embodiment, the IRES and second gene are in frame. In someembodiments, an adenovirus vector of the present invention furthercomprises E3.

Runaway infection is prevented due to the cell-specific requirements forviral replication. Without wishing to be bound by any particular theory,production of adenovirus proteins can serve to activate and/or stimulatethe immune system, either generally or specifically toward target cellsproducing adenoviral proteins which can be an important consideration inthe cancer context, where individuals are often moderately to severelyimmunocompromised.

In particular embodiments, the adenoviral vector may be areplication-competent target-cell specific adenoviral vector where thevector comprises an adenoviral gene. In one embodiment, the adenoviralgene is essential for replication and is under transcriptional controlof a target cell-specific TRE.

In certain embodiments, the adenoviral vector may be areplication-competent target-cell specific adenoviral vector wherein thegene essential for replication is an early gene. In other embodimentsthe gene essential for replication may be a late gene.

In preferred embodiments the gene essential for replication is E1A orE1B. In particular embodiments, the adenovirus comprises both E1A andE1B. In further embodiments, the gene essential for replication is E1Bwherein E1B has a deletion of part or all of the 19-kDa region.

In some embodiments, the adenovirus vector comprises co-transcribedfirst and second genes under transcriptional control of a heterologous,target cell-specific transcriptional regulatory element (TRE), whereinthe second gene is under translational control of an internal ribosomeentry site (IRES). In this embodiment, it is preferred that theendogenous promoter of the second gene be mutated or deleted and in oneembodiment, the IRES and second gene are in frame.

In some embodiments of the adenovirus vector, E1A has a mutation in ordeletion of its endogenous promoter. In some embodiments, E1B has amutation in or a deletion of its endogenous promoter. In someembodiments, E1A has a mutation in or deletion of its endogenousenhancer. In other embodiments, E1B has a deletion in part or all of the19-kDa region.

In particular preferred embodiments, the target cell specific adenoviralvector is specific for target cells including bladder, liver, prostate,breast, colorectal and melanoma cells.

In certain preferred embodiments, the adenoviral gene(s) essential forreplication is under the control of a TRE(s) such as, but not limited toPB-TRE, PSA-TRE, MUC-TRE, AFP-TRE, CEA-TRE, tyrosinase-TRE, hKLK2-TRE,and uroplakin-TRE, as described herein.

Illustrative adenoviral vectors are summarized in Table 4.

In one aspect of the present invention, the adenovirus vectors comprisean intergenic IRES element(s) which links the translation of two or moregenes, thereby removing any potential for homologous recombination basedon the presence of identical TREs in the vector. Adenovirus vectorscomprising an IRES are stable and in some embodiments provide betterspecificity than vectors not containing an IRES. Another advantage of anadenovirus vector comprising an intergenic IRES is that the use of anIRES rather than a second TRE may provide additional space in the vectorfor an additional gene(s) such as a therapeutic gene.

Thus, the adenovirus vectors comprising a second gene under control ofan IRES retain a high level of target cell specificity and remain stablein the target cell. Accordingly, in one aspect of the invention, theviral vectors disclosed herein comprise at least one IRES within amulticistronic transcript, wherein production of the multicistronictranscript is regulated by a heterologous, target cell-specific TRE. Foradenovirus vectors comprising a second gene under control of an IRES, itis preferred that the endogenous promoter of a gene under translationalcontrol of an IRES be deleted so that the endogenous promoter does notinterfere with transcription of the second gene. It is preferred thatthe second gene be in frame with the IRES if the IRES contains aninitiation codon. If an initiation codon, such as ATG, is present in theIRES, it is preferred that the initiation codon of the second gene isremoved and that the IRES and the second gene are in frame.Alternatively, if the IRES does not contain an initiation codon or ifthe initiation codon is removed from the IRES, the initiation codon ofthe second gene is used. In one embodiment, the adenovirus vectorscomprises the adenovirus essential genes, E1A and E1B genes, under thetranscriptional control of a heterologous, cell-specific TRE, and anIRES introduced between E1A and E1B. Thus, both E1A and E1B are undercommon transcriptional control, and translation of E1B coding region isobtained by virtue of the presence of the IRES. In one embodiment, E1Ahas its endogenous promoter deleted. In another embodiment, E1A has anendogenous enhancer deleted and in yet an additional embodiment, E1A hasits endogenous promoter deleted and E1A enhancer I deleted. In anotherembodiment, E1B has its endogenous promoter deleted. In yet furtherembodiments, E1B has a deletion of part or all of the 19-kDa region.

To provide cytotoxicity to target cells, one or more transgenes having acytotoxic effect may be present in the vector. Additionally, oralternatively, an adenovirus gene that contributes to cytotoxicityand/or cell death, such as the adenovirus death protein (ADP) gene, canbe included in the vector, optionally under the selectivetranscriptional control of a heterologous TRE and optionally under thetranslational control of an IRES.

The subject vectors can be used for a wide variety of purposes. Thepurpose will vary with the target cell. Suitable target cells arecharacterized by the transcriptional activation of the cell specifictranscriptional response element in the adenovirus vehicle. Thetranscription initiation region will usually be activated in less thanabout 5%, more usually less than about 1%, and desirably by less thanabout 0.1% of the cells in the host.

Transcriptional Response Elements (TREs)

The adenovirus vectors of the invention comprise target cell specificTREs which direct preferential expression of an operatively linked gene(or genes) in a particular target cell. A TRE can be tissue-specific,tumor-specific, developmental stage-specific, cell status specific,etc., depending on the type of cell present in the tissue or tumor.

Cell- and tissue-specific transcriptional regulatory elements, as wellas methods for their identification, isolation, characterization,genetic manipulation and use for regulation of operatively linked codingsequences, are well known in the art. A TRE can be derived from thetranscriptional regulatory sequences of a single gene, or sequences fromdifferent genes can be combined to produce a functional TRE. Acell-specific TRE is preferentially functional in a limited population(or type) of cells, e.g., prostate cells or liver cells. Accordingly, insome embodiments, the TRE used is preferentially functional in any ofthe following cell types: prostate; liver; breast; urothelial cells(bladder); colorectal; lung; ovarian; pancreas; stomach; and uterine. Inother embodiments, in accordance with cell status, the TRE is functionalin or during: low oxygen conditions (hypoxia); certain stages of cellcycle, such as S phase; elevated temperature; ionizing radiation.

As is known in the art, activity of TREs can be inducible. InducibleTREs generally exhibit low activity in the absence of inducer, and areup-regulated in the presence of inducer. Inducers include, for example,nucleic acids, polypeptides, small molecules, organic compounds and/orenvironmental conditions such as temperature, pressure or hypoxia.Inducible TREs may be preferred when expression is desired only atcertain times or at certain locations, or when it is desirable totitrate the level of expression using an inducing agent. For example,transcriptional activity from the PSA-TRE, PB-TRE and hKLK2-TRE isinducible by androgen, as described herein and in PCT/US98/04080.Accordingly, in one embodiment of the present invention, an adenovirusvector comprises an inducible heterologous TRE.

TRE multimers are also useful in the disclosed vectors. For example, aTRE can comprise a tandem series of at least two, at least three, atleast four, or at least five promoter fragments. Alternatively, a TREcan comprise one or more promoter regions along with one or moreenhancer regions. TRE multimers can also comprise promoter and/orenhancer sequences from different genes. The promoter and enhancercomponents of a TRE can be in any orientation with respect to each otherand can be in any orientation and/or any distance from the codingsequence of interest, as long as the desired cell-specifictranscriptional activity is obtained.

The disclosed vectors are designed such that replication ispreferentially enhanced in target cells in which the TRE(s) is (are)functional. More than one TRE can be present in a vector, as long as theTREs are functional in the same target cell. However, it is important tonote that a given TRE can be functional in more than one type of targetcell. For example, the CEA-TRE functions in, among other cell types,gastric cancer cells, colorectal cancer cells, pancreatic cancer cellsand lung cancer cells.

A TRE for use in the present vectors may or may not comprise a silencer.The presence of a silencer (i.e., a negative regulatory element known inthe art) can assist in shutting off transcription (and thus replication)in non-target cells. Thus, presence of a silencer can confer enhancedcell-specific vector replication by more effectively preventingreplication in non-target cells. Alternatively, lack of a silencer maystimulate replication in target cells, thus conferring enhanced targetcell-specificity.

As is readily appreciated by one skilled in the art, a TRE is apolynucleotide sequence, and, as such, can exhibit function over avariety of sequence permutations. Methods of nucleotide substitution,addition, and deletion are known in the art, and readily-availablefunctional assays (such as the CAT or luciferase reporter gene assay)allow one of ordinary skill to determine whether a sequence variantexhibits requisite cell-specific transcription regulatory function.Hence, functionally preserved variants of TREs, comprising nucleic acidsubstitutions, additions, and/or deletions, can be used in the vectorsdisclosed herein. Accordingly, variant TREs retain function in thetarget cell but need not exhibit maximal function. In fact, maximaltranscriptional activation activity of a TRE may not always be necessaryto achieve a desired result, and the level of induction afforded by afragment of a TRE may be sufficient for certain applications. Forexample, if used for treatment or palliation of a disease state,less-than-maximal responsiveness may be sufficient if, for example, thetarget cells are not especially virulent and/or the extent of disease isrelatively confined.

Certain base modifications may result in enhanced expression levelsand/or cell-specificity. For example, nucleic acid sequence deletions oradditions within a TRE can move transcription regulatory protein bindingsites closer or farther away from each other than they exist in theirnormal configuration, or rotate them so they are on opposite sides ofthe DNA helix, thereby altering spatial relationship among TRE-boundtranscription factors, resulting in a decrease or increase intranscription, as is known in the art. Thus, while not wishing to bebound by theory, the present disclosure contemplates the possibilitythat certain modifications of a TRE will result in modulated expressionlevels as directed by the TRE, including enhanced cell-specificity.Achievement of enhanced expression levels may be especially desirable inthe case of more aggressive forms of neoplastic growth, and/or when amore rapid and/or aggressive pattern of cell killing is warranted (forexample, in an immunocompromised individual).

Transcriptional activity directed by a TRE (including both inhibitionand enhancement) can be measured in a number of ways known in the art(and described in more detail below), but is generally measured bydetection and/or quantitation of mRNA and/or of a protein productencoded by the sequence under control of (i.e., operably linked to) aTRE.

As discussed herein, a TRE can be of varying lengths, and of varyingsequence composition. The size of a heterologous TRE will be determinedin part by the capacity of the viral vector, which in turn depends uponthe contemplated form of the vector (see infra). Generally minimal sizesare preferred for TREs, as this provides potential room for insertion ofother sequences which may be desirable, such as transgenes (discussedinfra) and/or additional regulatory sequences. In a preferredembodiment, such an additional regulatory sequence is an IRES. However,if no additional sequences are contemplated, or if, for example, anadenoviral vector will be maintained and delivered free of any viralpackaging constraints, larger TRE sequences can be used as long as theresultant adenoviral vector remains replication-competent.

In a preferred embodiment, a viral vector is an adenoviral vector. Anadenoviral vector can be packaged with extra sequences totaling up toabout 5% of the genome size, or approximately 1.8 kb, without requiringdeletion of viral sequences. If non-essential sequences are removed fromthe adenovirus genome, an additional 4.6 kb of insert can be tolerated(i.e., for a total insertion capacity of about 6.4 kb). Examples ofnon-essential adenoviral sequences that can be deleted are E3, and E4sequences other than those which encode E4 ORF6.

To minimize non-specific replication, endogenous (e.g., adenovirus) TREsare preferably removed from the vector. Besides facilitating targetcell-specific replication, removal of endogenous TREs also providesgreater insert capacity in a vector, which may be of special concern ifan adenoviral vector is to be packaged within a virus particle. Evenmore importantly, deletion of endogenous TREs prevents the possibilityof a recombination event whereby a heterologous TRE is deleted and theendogenous TRE assumes transcriptional control of its respectiveadenovirus coding sequences (thus allowing non-specific replication). Inone embodiment, an adenoviral vector is constructed such that theendogenous transcription control sequences of adenoviral genes aredeleted and replaced by one or more heterologous TREs. However,endogenous TREs can be maintained in the adenovirus vector(s), providedthat sufficient cell-specific replication preference is preserved. Theseembodiments are constructed by inserting heterologous TREs between anendogenous TRE and a replication gene coding segment. Requisitecell-specific replication preference is determined by conducting assaysthat compare replication of the adenovirus vector in a cell which allowsfunction of the heterologous TREs with replication in a cell which doesnot.

Generally, a TRE will increase replication of a vector in a target cellby at least about 2-fold, preferably at least about 5-fold, preferablyat least about 10-fold more preferably at least about 20-fold, morepreferably at least about 50-fold, more preferably at least about100-fold, more preferably at least about 200-fold, even more preferablyat least about 400- to about 500-fold, even more preferably at leastabout 1000-fold, compared to basal levels of replication in the absenceof a TRE. The acceptable differential can be determined empirically (bymeasurement of mRNA levels using, for example, RNA blot assays, RNaseprotection assays or other assays known in the art) and will depend uponthe anticipated use of the vector and/or the desired result.

Replication-competent adenovirus vectors directed at specific targetcells can be generated using TREs that are preferentially functional ina target cell. In one embodiment of the present invention, the targetcell is a tumor cell. Non-limiting examples of tumor cell-specificheterologous TREs, and their respective target cells, include: probasin(PB), target cell, prostate cancer (PCT/US98/04132); α-fetoprotein(AFP), target cell liver cancer (PCT/US98/04084); mucin-likeglycoprotein DF3 (MUC1), target cell, breast carcinoma (PCT/US98/04080);carcinoembryonic antigen (CEA), target cells, colorectal, gastric,pancreatic, breast, and lung cancers (PCT/US98/04133); plasminogenactivator urokinase (uPA) and its receptor gene, target cells, breast,colon, and liver cancers (PCT/US98/04080); E2F1 (cell cycle S-phasespecific promoter); target cell, tumors with disrupted retinoblastomagene function, and HER-2/neu (c-erbB2/neu), target cell, breast,ovarian, stomach, and lung cancers (PCT/US98/04080); tyrosinase, targetcell, melanoma cells as described herein and uroplakins, target cell,bladder cells as described herein. Methods for identification,isolation, characterization and utilization of additional targetcell-specific TREs are readily available to those of skill in the art.

In addition, tumor-specific TREs can be used in conjunction withtissue-specific TREs from the following exemplary genes (tissue in whichthe TREs are specifically functional are in parentheses): hypoxiaresponsive element, vascular endothelial growth factor receptor(endothelium), albumin (liver), factor VII (liver), fatty acid synthase(liver), Von Willebrand factor (brain endothelium), alpha-actin andmyosin heavy chain (both in smooth muscle), synthetase I (smallintestine) Na⁺—K⁺—Cl⁻ transporter (kidney). Additional tissue-specificTREs are known in the art.

In one embodiment of the present invention, a target cell-specific,heterologous TRE is tumor cell-specific. A vector can comprise a singletumor cell-specific TRE or multiple heterologous TREs which are tumorcell-specific and functional in the same cell. In another embodiment, avector comprises one or more heterologous TREs which are tumorcell-specific and additionally comprises one or more heterologous TREswhich are tissue specific, whereby all TREs are functional in the samecell.

Prostate-Specific TREs

In one embodiment, adenovirus vectors comprise heterologous TREs thatare prostate cell specific. For example, TREs that functionpreferentially in prostate cells and can be used to target adenovirusreplication to prostate neoplasia, include, but are not limited to, TREsderived from the prostate-specific antigen gene (PSA-TRE) (HendersonU.S. Pat. No. 5,698,443); the glandular kallikrein-1 gene (from thehuman gene, hKLK2-TRE) (PCT US98/16312), and the probasin gene (PB-TRE)(PCT/US98/04132). All three of these genes are preferentially expressedin prostate cells and their expression is androgen-inducible. Generally,expression of genes responsive to androgen induction is mediated by anandrogen receptor (AR).

Prostate-Specific Antigen (PSA)

PSA is synthesized exclusively in prostatic epithelial cells and issynthesized in these cells whether they are normal, hyperplastic, ormalignant. This tissue-specific expression of PSA has made it anexcellent biomarker for benign prostatic hyperplasia (BPH) and prostaticcarcinoma (CaP). Normal serum levels of PSA are typically below 5 ng/ml,with elevated levels indicative of BPH or CaP. Lundwall et al. (1987)FEBS Lett. 214:317; Lundwall (1989) Biochem. Biophys. Res. Comm.161:1151; and Riegmann et al. (1991) Molec. Endocrin. 5:1921.

The region of the PSA gene that provides androgen-dependent cellspecificity, particularly in prostate cells, involves approximately 6.0kilobases (kb). Schuur et al. (1996) J. Biol. Chem. 271:7043-7051. Anenhancer region of approximately 1.5 kb in humans is located between nt−5322 and nt −3739, relative to the transcription start site of the PSAgene. Within these enhancer sequences is an androgen response element(ARE) a sequence which binds androgen receptor. The sequence coordinatesof the PSA promoter are from about nt −540 to nt +8 relative to thetranscription start site. Juxtapositioning of the enhancer and promoteryields a fully functional, minimal prostate-specific TRE (PSA-TRE).Other portions of this approximately 6.0 kb region of the PSA gene canbe used in the vectors described herein, as long as requisitefunctionality is maintained.

Human Glandular Kallikrein (hKLK2)

Human glandular kallikrein (hKLK2, encoding the hK2 protein) isexpressed exclusively in the prostate and its expression is up-regulatedby androgens, primarily through transcriptional activation. Wolf et al.(1992) Molec. Endocrinol. 6:753-762; Morris (1989) Clin. Exp. Pharm.Physiol. 16:345-351; Qui et al. (1990) J. Urol. 144:1550-1556; and Younget al. (1992) Biochem. 31:818-824. The levels of hK2 found in varioustumors and in the serum of patients with prostate cancer indicate thathK2 antigen may be a significant marker for prostate cancer.Charlesworth et al. (1997) Urology 49:487-493. Expression of hK2 hasbeen detected in each of 257 radical prostatectomy specimens analyzed.Darson et al. (1997) Urology 49:857-862. The intensity and extent of hK2expression, detected using specific antibodies, was observed to increasefrom benign epithelium to high-grade prostatic intraepithelial neoplasia(PIN) and adenocarcinoma.

The activity of the hKLK2 promoter has been described and a region up tont −2256 relative to the transcription start site was previouslydisclosed. Schedlich et al. (1987) DNA 6:429-437. The hKLK2 promoter isandrogen responsive and, in plasmid constructs wherein the promoteralone controls the expression of a reporter gene, expression of thereporter gene is increased approximately 10-fold in the presence ofandrogen. Murtha et al. (1993) Biochem. 32:6459-6464. hKLK2 enhanceractivity is found within a polynucleotide sequence approximately nt−12,014 to nt −2257 relative to the start of transcription and, whenthis sequence is operably linked to an hKLK2 promoter and a reportergene, transcription of operably-linked sequences in prostate cellsincreases in the presence of androgen to levels approximately 30-fold toapproximately 100-fold greater than the level of transcription in theabsence of androgen. This induction is generally independent of theorientation and position of the enhancer sequences. Enhancer activityhas also been demonstrated in the following regions (all relative to thetranscription start site): about nt −3993 to about nt −3643, about nt−4814 to about nt −3643, about nt −5155 to about nt −3387, about nt−6038 to about nt −2394.

Thus, a hKLK2 enhancer can be operably linked to an hKLK2 promoter or aheterologous promoter to form a hKLK2 transcriptional regulatory element(hKLK2-TRE). A hKLK2-TRE can then be operably linked to a heterologouspolynucleotide to confer hKLK2-TRE-specific transcriptional regulationon the linked gene, thus increasing its expression.

Probasin

The rat probasin (PB) gene encodes an androgen and zinc-regulatedprotein first characterized in the dorsolateral prostate of the rat.Dodd et al. (1983) J. Biol. Chem. 258:10731-10737; Matusik et al. (1986)Biochem. Cell. Biol. 64:601-607; and Sweetland et al. (1988) Mol. Cell.Biochem. 84:3-15. The dorsolateral lobes of the murine prostate areconsidered the most homologous to the peripheral zone of the humanprostate, where approximately 68% of human prostate cancers are thoughtto originate.

A PB-TRE has been shown to exist in an approximately 0.5 kb fragment ofsequence upstream of the probasin coding sequence, from about nt −426 toabout nt +28 relative to the transcription start site. This minimalpromoter sequence from the PB gene appears to provide sufficientinformation to direct prostate-specific developmental- andhormone-regulated expression of an operably linked heterologous gene intransgenic mice. Greenberg et al. (1994) Mol. Endocrinol. 8:230-239.

Alpha-Fetoprotein

α-fetoprotein (AFP) is an oncofetal protein, the expression of which isprimarily restricted to developing tissues of endodermal origin (yolksac, fetal liver, and gut), although the level of its expression variesgreatly depending on the tissue and the developmental stage. AFP is ofclinical interest because the serum concentration of AFP is elevated ina majority of hepatoma patients, with high levels of AFP found inpatients with advanced disease. High serum AFP levels in patients appearto be due to AFP expression in hepatocellular carcinoma (HCC), but notin surrounding normal liver. Thus, expression of the AFP gene appears tobe characteristic of hepatoma cells. An AFP-TRE is described in forexample PCT/US98/04084.

According to published reports, the AFP-TRE is responsive to cellularproteins (transcription factors and/or co-factor(s)) associated withAFP-producing cells, such as AFP-binding protein (see, for example, U.S.Pat. No. 5,302,698) and comprises at least a portion of an AFP promoterand/or an AFP enhancer. Cell-specific TREs from the AFP gene have beenidentified. For example, the cloning and characterization of humanAFP-specific enhancer activity is described in Watanabe et al. (1987) J.Biol. Chem. 262:4812-4818. A 5′ AFP regulatory region (containing thepromoter, putative silencer, and enhancer) is contained withinapproximately 5 kb upstream from the transcription start site.

Within the AFP regulatory region, a human AFP enhancer region is locatedbetween about nt −3954 and about nt −3335, relative to the transcriptionstart site of the AFP gene. The human AFP promoter encompasses a regionfrom about nt −174 to about nt +29. Juxtapositioning of these twogenetic elements, yields a fully functional AFP-TRE. Ido et al. (1995)Cancer Res. 55:3105-3109 describe a 259 bp promoter fragment (nt −230 tont +29) that is specific for expression in HCC cells. The AFP enhancer,located between nt −3954 and nt −3335 relative to the transcriptionstart site, contains two regions, denoted A and B. The promoter regioncontains typical TATA and CAAT boxes. Preferably, the AFP-TRE containsat least one enhancer region. More preferably, the AFP-TRE contains bothenhancer regions.

Suitable target cells for vectors containing AFP-TREs are any cell typethat allow an AFP-TRE to function. Preferred are cells that express orproduce AFP, including, but not limited to, tumor cells expressing AFP.Examples of such cells are hepatocellular carcinoma (HCC) cells, gonadaland other germ cell tumors (especially endodermal sinus tumors), braintumor cells, ovarian tumor cells, acinar cell carcinoma of the pancreas(Kawamoto et al. (1992) Hepatogastroenterology 39:282-286), primary gallbladder tumor (Katsuragi et al. (1989) Rinsko Hoshasen 34:371-374),uterine endometrial adenocarcinoma cells (Koyama et al. (1996) Jpn. J.Cancer Res. 87:612-617), and any metastases of the foregoing (which canoccur in lung, adrenal gland, bone marrow, and/or spleen). In somecases, metastatic disease to the liver from certain pancreatic andstomach cancers produce AFP Especially preferred as target cells for anAFP-TRE are hepatocellular carcinoma cells and any of their metastases.

AFP production can be measured (and hence AFP-producing cells can beidentified) using immunoassays standard in the art, such as RIA, ELISAor protein immunoblotting (Western blots) to determine levels of AFPprotein production; and/or RNA blotting (Northern blots) to determineAFP mRNA levels. Alternatively, such cells can be identified and/orcharacterized by their ability to activate transcriptionally an AFP-TRE(i.e., allow an AFP-TRE to function).

See also co-owned PCT WO98/39465 regarding AFP-TREs. As described inmore detail therein, an AFP-TRE can comprise any number ofconfigurations, including, but not limited to, an AFP promoter; an AFPenhancer; an AFP promoter and an AFP enhancer; an AFP promoter and aheterologous enhancer; a heterologous promoter and an AFP enhancer, andmultimers of the foregoing. The promoter and enhancer components of anAFP-TRE can be in any orientation and/or distance from the codingsequence of interest, as long as the desired AFP cell-specifictranscriptional activity is obtained. An adenovirus vector of thepresent invention can comprise an AFP-TRE endogenous silencer element orthe AFP-TRE endogenous silencer element can be deleted.

Urokinase Plasminogen Activator

The protein urokinase plasminogen activator (uPA) and its cell surfacereceptor, urokinase plasminogen activator receptor (uPAR), are expressedin many of the most frequently-occurring neoplasms and appear torepresent important proteins in cancer metastasis. Both proteins areimplicated in breast, colon, prostate, liver, renal, lung and ovariancancer. Sequence elements that regulate uPA and uPAR transcription havebeen extensively studied. Riccio et al. (1985) Nucleic Acids Res.13:2759-2771; Cannio et al. (1991) Nucleic Acids Res. 19:2303-2308.

Carcinoembryonic Antigen (CEA)

CEA is a 180,000 Dalton, tumor-associated, glycoprotein antigen presenton endodermally-derived neoplasms of the gastrointestinal tract, such ascolorectal, gastric (stomach) and pancreatic cancer, as well as otheradenocarcinomas such as breast and lung cancers. CEA is of clinicalinterest because circulating CEA can be detected in the great majorityof patients with CEA-positive tumors. In lung cancer, about 50% of totalcases have circulating CEA, with high concentrations of CEA (greaterthan 20 ng/ml) often detected in adenocarcinomas. Approximately 50% ofpatients with gastric carcinoma are serologically positive for CEA.

The 5′-flanking sequence of the CEA gene has been shown to confercell-specific activity. The CEA promoter region, approximately the first424 nucleotides upstream of the transcriptional start site in the 5′flanking region of the gene, was shown to confer cell-specific activityby virtue of providing higher promoter activity in CEA-producing cellsthan in non-producing HeLa cells. Schrewe et al. (1990) Mol. Cell. Biol.10:2738-2748. In addition, cell-specific enhancer regions have beenfound. See PCT/GB/02546 The CEA promoter, putative silencer, andenhancer elements appears to be contained within a region that extendsapproximately 14.5 kb upstream from the transcription start site.Richards et al. (1995); PCT/GB/02546. Further characterization of the5′-flanking region of the CEA gene by Richards et al. (1995) supraindicated that two upstream regions (one between about −13.6 and about−10.7 kb, and the other between about −6.1 and about −4.0 kb), whenlinked to the multimerized promoter, resulted in high-level andselective expression of a reporter construct in CEA-producing LoVo andSW1463 cells. Richards et al. (1995) supra also localized the promoterregion between about nt −90 and about nt +69 relative to thetranscriptional start site, with the region between about nt −41 andabout nt −18 being essential for expression. PCT/GB/02546 describes aseries of 5′-flanking CEA fragments which confer cell-specific activity,including fragments comprising the following sequences: about nt −299 toabout nt +69; about nt −90 to about nt +69; nt −14,500 to nt −10,600; nt−13,600 to nt −10,600; and nt −6100 to nt −3800, with all coordinatesbeing relative to the transcriptional start point. In addition,cell-specific transcription activity is conferred on an operably linkedgene by the CEA fragment from nt 402 to nt +69.

CEA-TREs for use in the vectors disclosed herein are derived frommammalian cells, including, but not limited to, human cells. Thus, anyof the CEA-TREs can be used as long as the requisite desiredfunctionality is displayed by the vector.

Mucin

The protein product of the MUC1 gene (known as mucin, MUC1 protein;episialin; polymorphic epithelial mucin or PEM; EMA; DF3 antigen; NPGP;PAS-O; or CA15.3 antigen) is normally expressed mainly at the apicalsurface of epithelial cells lining the glands or ducts of the stomach,pancreas, lungs, trachea, kidney, uterus, salivary glands, and mammaryglands. Zotter et al. (1988) Cancer Rev. 11-12:55-101; and Girling etal. (1989) Int. J. Cancer 43:1072-1076. However, mucin is overexpressedin 75-90% of human breast carcinomas. Kufe et al. (1984) Hybridoma3:223-232. For reviews, see Hilkens (1988) Cancer Rev. 11-12:25-54; andTaylor-Papadimitriou, et al. (1990) J. Nucl. Med. Allied Sci.34:144-150. Mucin protein expression correlates with the degree ofbreast tumor differentiation. Lundy et al. (1985) Breast Cancer Res.Treat. 5:269-276.

Overexpression of the MUC1 gene in human breast carcinoma cells MCF-7and ZR-75-1 appears to occur at the transcriptional level. Kufe et al.(1984) supra; Kovarik (1993) J. Biol. Chem. 268:9917-9926; and Abe etal. (1990) J. Cell. Physiol. 143:226-231. The regulatory sequences ofthe MUC1 gene have been cloned, including the approximately 0.9 kbupstream of the transcription start site which contains a TRE thatappears to be involved in cell-specific transcription. Abe et al. (1993)Proc. Natl. Acad. Sci. USA 90:282-286; Kovarik et al. (1993) supra; andKovarik et al. (1996) J. Biol. Chem. 271:18140-18147.

MUC1-TREs are derived from mammalian cells, including but not limitedto, human cells. Preferably, the MUC1-TRE is human. In one embodiment,the MUC1-TRE contains the entire 0.9 kb 5′ flanking sequence of the MUC1gene. In other embodiments, MUC1-TREs comprise the following sequences(relative to the transcription start site of the MUC1 gene)operably-linked to a promoter: about nt −725 to about nt +31, about nt−743 to about nt +33, about nt −750 to about nt +33, and about nt −598to about nt +485.

c-erbB2/HER-2/neu

The c-erbB2/neu gene (HER-2/neu or HER) is a transforming gene thatencodes a 185 kD epidermal growth factor receptor-related transmembraneglycoprotein. In humans, the c-erbB2/neu protein is expressed duringfetal development and, in adults, the protein is weakly detectable (byimmunohistochemistry) in the epithelium of many normal tissues.Amplification and/or over-expression of the c-erbB2/neu gene has beenassociated with many human cancers, including breast, ovarian, uterine,prostate, stomach and lung cancers. The clinical consequences ofoverexpression of the c-erbB2/neu protein have been best studied inbreast and ovarian cancer. c-erbB2/neu protein over-expression occurs in20 to 40% of intraductal carcinomas of the breast and 30% of ovariancancers, and is associated with a poor prognosis in subcategories ofboth diseases.

Human, rat and mouse c-erbB2/neu TREs have been identified and shown toconfer transcriptional activity specific to c-erbB2/neu-expressingcells. Tal et al. (1987) Mol. Cell. Biol. 7:2597-2601; Hudson et al.(1990) J. Biol. Chem. 265:4389-4393; Grooteclaes et al. (1994) CancerRes. 54:4193-4199; Ishii et al. (1987) Proc. Natl. Acad. Sci. USA84:4374-4378; and Scott et al. (1994) J. Biol. Chem. 269:19848-19858.

Melanocyte-Specific TRE

It has been shown that some genes which encode melanoma proteins arefrequently expressed in melanoma/melanocytes, but silent in the majorityof normal tissues. A variety of melanocyte-specific TRE are known, areresponsive to cellular proteins (transcription factors and/orco-factor(s)) associated with melanocytes, and comprise at least aportion of a melanocyte-specific promoter and/or a melanocyte-specificenhancer. Known transcription factors that control expression of one ormore melanocyte-specific genes include the microphthalmia associatedtranscription factor MITF. Yasumoto et al. (1997) J. Biol. Chem.272:503-509. Other transcription factors that control expression of oneor more melanocyte specific genes include MART-1/Melan-A, gp100, TRP-1and TRP-2.

Methods are described herein for measuring the activity of amelanocyte-specific TRE and thus for determining whether a given cellallows a melanocyte-specific TRE to function.

The melanocyte-specific TREs used in this invention are derived frommammalian cells, including but not limited to, human, rat, and mouse.Any melanocyte-specific TREs may be used in the adenoviral vectors ofthe invention. Rodent and human 5′ flanking sequences from genesexpressed specifically or preferentially in melanoma cells have beendescribed in the literature and are thus made available for practice ofthis invention and need not be described in detail herein. The followingare some examples of melanocyte-specific TREs which can be used. Apromoter and other control elements in the human tyrosinase gene 5′flanking region have been described and sequences have been deposited asGenBank Accession Nos. X16073 and D10751. Kikuchi et al. (1989) Biochim.Biophys. Acta 1009:283-286; and Shibata et al. (1992) J. Biol. Chem.267:20584-20588. A cis-acting element has been defined that enhancesmelanocyte-specific expression of human tyrosinase gene. This elementcomprises a 20-bp sequence known as tyrosinase distal element (TDE),contains a CATGTG motif, and lies at positions about −1874 to about−1835 relative to the human tyrosinase gene transcription start site.Yasumoto et al. (1994) Mol. Cell. Biol. 14:8058-8070. A promoter regioncomprising sequences from about −209 to +61 of the human tyrosinase genewas found to direct melanocyte-specific expression. Shibata (1992).Similarly, the mouse tyrosinase 5′ flanking region has been analyzed anda sequence deposited as GenBank Accession Nos. D00439 and X51743.Klüppel et al. (1991) Proc. Natl. Acad. Sci. USA 88:3777-3788. A minimalpromoter has been identified for the mouse TRP-1 gene, and was reportedto encompass nucleotides 44 to +107 relative to the transcription startsite. Lowings et al. (1992) Mol. Cell. Biol. 12:3653-3662. Tworegulatory regions required for melanocyte-specific expression of thehuman TRP-2 gene have been identified. Yokoyama et al. (1994) J. Biol.Chem. 269:27080-27087. A human MART-1 promoter region has been describedand deposited as GenBank Accession No. U55231. Melanocyte-specificpromoter activity was found in a 233-bp fragment of the human MART-1gene 5′ flanking region. Butterfield et al. (1997) Gene 191:129-134. Abasic-helix-loop-helix/leucine zipper-containing transcription factor,MITF (microphthalmia associated transcription factor) was reported to beinvolved in transcriptional activation of tyrosinase and TRP-1 genes.Yasumoto et al. (1997) J. Biol. Chem. 272:503-509.

In some embodiments, a melanocyte-specific TRE comprises sequencesderived from the 5′ flanking region of a human tyrosinase gene depictedin Table 14. In some of these embodiments, the melanocyte-specific TREcomprises tyrosinase nucleotides from about −231 to about +65 relativeto the transcription start site (from about nucleotide 244 to aboutnucleotide 546 of SEQ ID NO:______) and may further comprise nucleotidesfrom about −1956 to about −1716 relative to the human tyrosinasetranscription start site (from about nucleotide 6 to about nucleotide−243 of SEQ ID NO:______). A tyrosinase TRE can comprise nucleotidesfrom about −231 to about +65 juxtaposed to nucleotides from about −1956to about −1716. It has been reported that nucleotides from about −1956to about −1716 relative to the human tyrosinase transcription start sitecan confer melanocyte-specific expression of an operably linked reportergene with either a homologous or a heterologous promoter. Accordingly,in some embodiments, a melanocyte-specific TRE comprises nucleotidesfrom about −1956 to about −1716 operably linked to a heterologouspromoter.

A melanocyte-specific TRE can also comprise multimers. For example, amelanocyte-specific TRE can comprise a tandem series of at least two, atleast three, at least four, or at least five tyrosinase promoterfragments. Alternatively, a melanocyte-specific TRE could have one ormore tyrosinase promoter regions along with one or more tyrosinaseenhancer regions. These multimers may also contain heterologous promoterand/or enhancer sequences.

Cell Status-Specific TREs

Cell status-specific TREs for use in the adenoviral vectors of thepresent invention can be derived from any species, preferably a mammal.A number of genes have been described which are expressed in responseto, or in association with, a cell status. Any of these cellstatus-associated genes may be used to generate a cell status-specificTRE.

An example of a cell status is cell cycle. An exemplary gene whoseexpression is associated with cell cycle is E2F-1, a ubiquitouslyexpressed, growth-regulated gene, which exhibits peak transcriptionalactivity in S phase. Johnson et al. (1994) Genes Dev. 8:1514-1525. TheRB protein, as well as other members of the RB family, form specificcomplexes with E2F-1, thereby inhibiting its ability to activatetranscription. Thus, E2F-1-responsive promoters are down-regulated byRB. Many tumor cells have disrupted RB function, which can lead tode-repression of E2F-1-responsive promoters, and, in turn, de-regulatedcell division.

Accordingly, in one embodiment, the invention provides an E3-containingadenoviral vector in which an adenoviral gene (preferably a genenecessary for replication) is under transcriptional control of a cellstatus-specific TRE, wherein the cell status-specific TRE comprises acell cycle-activated TRE. In one embodiment, the cell cycle-activatedTRE is an E2F1 TRE.

Another group of genes that are regulated by cell status are those whoseexpression is increased in response to hypoxic conditions. Bunn andPoyton (1996) Physiol. Rev. 76:839-885; Dachs and Stratford (1996) Br.J. Cancer 74:5126-5132; Guillemin and Krasnow (1997) Cell 89:9-12. Manytumors have insufficient blood supply, due in part to the fact thattumor cells typically grow faster than the endothelial cells that makeup the blood vessels, resulting in areas of hypoxia in the tumor.Folkman (1989) J. Natl. Cancer Inst. 82:4-6; and Kallinowski (1996) TheCancer J. 9:3740. An important mediator of hypoxic responses is thetranscriptional complex HIF-1, or hypoxia inducible factor-1, whichinteracts with a hypoxia-responsive element (HRE) in the regulatoryregions of several genes, including vascular endothelial growth factor,and several genes encoding glycolytic enzymes, including enolase-1.Murine HRE sequences have been identified and characterized. Firth etal. (1994) Proc. Natl. Acad. Sci. USA 91:6496-6500. An HRE from a ratenolase-1 promoter is described in Jiang et al. (1997) Cancer Res.57:5328-5335. An HRE from a rat enolase-1 promoter is depicted in Table14.

Accordingly, in one embodiment, an adenovirus vector comprises anadenovirus gene, preferably an adenoviral gene essential forreplication, under transcriptional control of a cell status-specific TREcomprising an HRE. In one embodiment, the cell status-specific TREcomprises the HRE depicted in Table 14.

Other cell status-specific TREs include heat-inducible (i.e., heatshock) promoters, and promoters responsive to radiation exposure,including ionizing radiation and UV radiation. For example, the promoterregion of the early growth response-1 (Egr-1) gene contains anelement(s) inducible by ionizing radiation. Hallahan et al. (1995) Nat.Med. 1:786-791; and Tsai-Morris et al. (1988) Nucl. Acids. Res.16:8835-8846. Heat-inducible promoters, including heat-inducibleelements, have been described. See, for example Welsh (1990) in “StressProteins in Biology and Medicine”, Morimoto, Tisseres, and Georgopoulos,eds. Cold Spring Harbor Laboratory Press; and Perisic et al. (1989) Cell59:797-806. Accordingly, in some embodiments, the cell status-specificTRE comprises an element(s) responsive to ionizing radiation. In oneembodiment, this TRE comprises a 5′ flanking sequence of an Egr-1 gene.In other embodiments, the cell status-specific TRE comprises a heatshock responsive element.

The cell status-specific TREs listed above are provided as non-limitingexamples of TREs that would function in the instant invention.Additional cell status-specific TREs are known in the art, as aremethods to identify and test cell status specificity of suspected cellstatus-specific TREs.

Urothelial Cell-Specific TREs

Any urothelial cell-specific TRE may be used in the adenoviral vectorsof the invention. A number of urothelial cell-specific proteins havebeen described, among which are the uroplakins. Uroplakins (UP),including UPIa and UPIb (27 and 28 kDa, respectively), UPII (15 kDa),and UPIII (47 kDa), are members of a group of integral membrane proteinsthat are major proteins of urothelial plaques. These plaques cover alarge portion of the apical surface of mammalian urothelium and may playa role as a permeability barrier and/or as a physical stabilizer of theurothelial apical surface. Wu et al. (1994) J. Biol. Chem.269:13716-13724. UPs are bladder-specific proteins, and are expressed ona significant proportion of urothelial-derived tumors, including about88% of transitional cell carcinomas. Moll et al. (1995) Am. J. Pathol.147:1383-1397; and Wu et al. (1998) Cancer Res. 58:1291-1297. Thecontrol of the expression of the human UPII has been studied, and a3.6-kb region upstream of the mouse UPI gene has been identified whichcan confer urothelial-specific transcription on heterologous genes (Linet al. (1995) Proc. Natl. Acad. Sci. USA 92:679-683).

Preferred urothelial cell-specific TREs include TREs derived from theuroplakins UPIa, UPIb, UPII, and UPIII, as well as urohingin. Auroplakin TRE may be from any species, depending on the intended use ofthe adenovirus, as well as the requisite functionality is exhibited inthe target or host cell. Significantly, adenovirus constructs comprisinga urothelial cell-specific TREs have observed that such constructs arecapable of selectively replicating in urothelial cells as opposed tosmooth muscle cells, which adjoin urothelial cells in the bladder.

Uroplakin

Urothelial-specific TREs derived from the hUPII gene are describedherein. Accordingly, in some embodiments, an adenovirus vector of theinvention comprises an adenovirus gene, preferably an adenoviral geneessential for replication, under transcriptional control of a urothelialcell-specific TRE which comprises the 2.2 kb sequence from the 5′flanking region of hUPII gene, as shown in Table 14. In otherembodiments, an adenovirus vector of the invention comprises anadenovirus gene, preferably an adenoviral gene essential forreplication, under transcriptional control of a urothelial cell-specificTRE which comprises a 1.8 kb sequence from the 5′ flanking region ofhUPII gene, from nucleotides 430 to 2239 as shown in Table 14. In otherembodiments, the urothelial cell-specific TRE comprises a functionalportion of the 2.2 kb sequence depicted in Table 14, or a functionalportion of the 1.8 kb sequence of nucleotides 430 to 2239 of thesequence depicted in Table 14, such as a fragment of 2000 bp or less,1500 bp or less, or 1000 bp or less, 600 bp less, or at least 200 bpwhich includes the 200 bp fragment of the hUPII 5′-flanking region.

A 3.6 kb 5′-flanking sequence located from the mouse UPII (mUPII) genewhich confers urothelial cell-specific transcription on heterologousgenes is one urothelial cell-specific TRE useful in vectors of theinstant invention (Table 14). Smaller TREs (i.e., 3500 bp or less, morepreferably less than about 2000 bp, 1500 bp, or 1000 bp) are preferred.Smaller TREs derived from the mUPII 3.6 kb fragment are one group ofpreferred urothelial cell-specific TREs. In particular, Inventors haveidentified an approximately 600 bp fragment from the 5′ flanking DNA ofthe mUPII gene, which contains 540 bp of 5′ untranslated region (UTR) ofthe mUPII gene, that confers urothelial cell-specific expression onheterologous genes.

Accordingly, in some embodiments, an adenovirus vector comprises anadenovirus gene, preferably an adenoviral gene essential forreplication, under transcriptional control of a urothelial cell-specificTRE which comprises the 3.6 kb sequence from the 5′ flanking region ofmouse UPII gene, as shown in Table 14. In other embodiments, theurothelial cell-specific TRE comprises a functional portion of the 3.6kb sequence depicted in Table 14, such as a fragment of 3500 bp or less,2000 bp or less, 1500 bp or less, or 1000 bp or less which includes the540 bp fragment of 5′ UTR. The urothelial cell-specific TRE may also bea sequence which is substantially identical to the 3.6 kb mUPII5′-flanking region or any of the described fragments thereof.

As an example of how urothelial cell-specific TRE activity can bedetermined, a polynucleotide sequence or set of such sequences can begenerated using methods known in the art, such as chemical synthesis,site-directed mutagenesis, PCR, and/or recombinant methods. Thesequence(s) to be tested is inserted into a vector containing anappropriate reporter gene, including, but not limited to,chloramphenicol acetyl transferase (CAT), β-galactosidase (encoded bythe lacZ gene), luciferase (encoded by the luc gene), a greenfluorescent protein, alkaline phosphatase, and horse radish peroxidase.Such vectors and assays are readily available, from, inter alia,commercial sources. Plasmids thus constructed are transfected into asuitable host cell to test for expression of the reporter gene ascontrolled by the putative target cell-specific TRE using transfectionmethods known in the art, such as calcium phosphate precipitation,electroporation, liposomes (lipofection) and DEAE dextran. Suitable hostcells include any urothelial cell type, including but not limited to,KU-1, MYP3 (a non-tumorigenic rat urothelial cell line), 804G (ratbladder carcinoma cell line), cultured human urothelial cells (HUC),HCV-29, UM-UC-3, SW780, RT4, HL60, KG-1, and KG-1A. Non-urothelialcells, such as LNCaP, HBL-100, HLF, HLE, 3T3, Hep3B, HuH7, CADO-LC9, andHeLa are used as a control. Results are obtained by measuring the levelof expression of the reporter gene using standard assays. Comparison ofexpression between urothelial cells and control indicates presence orabsence of transcriptional activation.

Comparisons between or among various urothelial cell-specific TREs canbe assessed by measuring and comparing levels of expression within asingle urothelial cell line. It is understood that absolutetranscriptional activity of a urothelial cell-specific TRE will dependon several factors, such as the nature of the target cell, delivery modeand form of the urothelial cell-specific TRE, and the coding sequencethat is to be selectively transcriptionally activated. To compensate forvarious plasmid sizes used, activities can be expressed as relativeactivity per mole of transfected plasmid. Alternatively, the level oftranscription (i.e., mRNA) can be measured using standard Northernanalysis and hybridization techniques. Levels of transfection (i.e.,transfection efficiencies) are measured by co-transfecting a plasmidencoding a different reporter gene under control of a different TRE,such as the CMV immediate early promoter. This analysis can alsoindicate negative regulatory regions, i.e., silencers.

Alternatively a putative urothelial cell-specific TRE can be assessedfor its ability to confer adenoviral replication preference for cellsthat allow a urothelial cell-specific TRE to function. For this assay,constructs containing an adenovirus gene essential to replicationoperatively linked to a putative urothelial cell-specific TRE aretransfected into urothelial cells. Viral replication in those cells iscompared, for example, to viral replication by wild type adenovirus inthose cells and/or viral replication by the construct in non-urothelialcells.

TRE Configurations

A TRE as used in the present invention can be present in a variety ofconfigurations. A TRE can comprise multimers. For example, a TRE cancomprise a tandem series of at least two, at least three, at least four,or at least five target cell-specific TREs. These multimers may alsocontain heterologous promoter and/or enhancer sequences.

Optionally, a transcriptional terminator or transcriptional “silencer”can be placed upstream of the target cell-specific TRE, thus preventingunwanted read-through transcription of the coding segment undertranscriptional control of the target cell-specific TRE. Also,optionally, the endogenous promoter of the coding segment to be placedunder transcriptional control of the target cell-specific TRE can bedeleted.

A target cell-specific TRE may or may not lack a silencer. The presenceof a silencer (i.e., a negative regulatory element) may assist inshutting off transcription (and thus replication) in non-permissivecells (i.e., a non-target cell). Thus, presence of a silencer may conferenhanced target cell-specific replication by more effectively preventingadenoviral vector replication in non-target cells. Alternatively, lackof a silencer may assist in effecting replication in target cells, thusconferring enhanced target cell-specific replication due to moreeffective replication in target cells.

It is also understood that the invention includes a target cell-specificTRE regulating the transcription of a bicistronic mRNA in whichtranslation of the second mRNA is associated by an IRES. An adenovirusvector may further include an additional heterologous TRE which may ormay not be operably linked to the same gene(s) as the targetcell-specific TRE. For example a TRE (such as a cell type-specific orcell status-specific TRE) may be juxtaposed to a second type oftarget-cell-specific TRE. “Juxtaposed” means a target cell-specific TREand a second TRE transcriptionally control the same gene. For theseembodiments, the target cell-specific TRE and the second TRE may be inany of a number of configurations, including, but not limited to, (a)next to each other (i.e., abutting); (b) both 5′ to the gene that istranscriptionally controlled (i.e., may have intervening sequencesbetween them); (c) one TRE 5′ and the other TRE 3′ to the gene.

As is readily appreciated by one skilled in the art, a targetcell-specific TRE is a polynucleotide sequence, and, as such, canexhibit function over a variety of sequence permutations. Methods ofnucleotide substitution, addition, and deletion are known in the art,and readily available functional assays (such as the CAT or luciferasereporter gene assay) allow one of ordinary skill to determine whether asequence variant exhibits requisite target cell-specific transcriptionfunction. Hence, the invention also includes functionally-preservedvariants of the TRE nucleic acid sequences disclosed herein, whichinclude nucleic acid substitutions, additions, and/or deletions. Thevariants of the sequences disclosed herein may be 80%, 85%, 90%, 95%,98%, 99% or more identical, as measured by, for example, ALIGN Plus(Scientific and Educational Software, Pennsylvania), preferably usingefault parameters, which are as follows: mismatch=2; open gap=0; extendgap=2 to any of the urothelial cell-specific TRE sequences disclosedherein. Variants of target cell-specific TRE sequences may alsohybridize at high stringency, that is at 68° C. and 0.1×SSC, to any ofthe target cell-specific TRE sequences disclosed herein.

In terms of hybridization conditions, the higher the sequence identityrequired, the more stringent are the hybridization conditions if suchsequences are determined by their ability to hybridize to a sequence ofa TRE disclosed herein. Accordingly, the invention also includespolynucleotides that are able to hybridize to a sequence comprising atleast about 15 contiguous nucleotides (or more, such as about 25, 35,50, 75 or 100 contiguous nucleotides) of a sequence of a TRE disclosedherein. The hybridization conditions would be stringent, i.e., 80° C.(or higher temperature) and 6M SSC (or less concentrated SSC). Anotherset of stringent hybridization conditions is 68° C. and 0.1×SSC. Fordiscussion regarding hybridization reactions, see below.

Hybridization reactions can be performed under conditions of different“stringency”. Conditions that increase stringency of a hybridizationreaction of widely known and published in the art. See, for example,Sambrook et al. (1989) at page 7.52. Examples of relevant conditionsinclude (in order of increasing stringency): incubation temperatures of25° C., 37° C., 50° C. and 68° C.; buffer concentrations of 10×SSC,6×SSC, 1×SSC, 0.1×SSC (where SSC is 0.15 M NaCl and 15 mM citratebuffer) and their equivalents using other buffer systems; formamideconcentrations of 0%, 25%, 50%, and 75%; incubation times from 5 minutesto 24 hours; 1, 2, or more washing steps; wash incubation times of 1, 2,or 15 minutes; and wash solutions of 6×SSC, 1×SSC, 0.1×SSC, or deionizedwater. An exemplary set of stringent hybridization conditions is 68° C.and 0.1×SSC.

“T_(m)” is the temperature in degrees Celcius at which 50% of apolynucleotide duplex made of complementary strands hydrogen bonded inanti-parallel direction by Watson-Crick base pairing dissociates intosingle strands under conditions of the experiment. T_(m) may bepredicted according to a standard formula, such as:T _(m)=81.5+16.6 log[X ⁺]+0.41(% G/C)−0.61(% F)−600/Lwhere [X⁺] is the cation concentration (usually sodium ion, Na⁺) inmol/L; (% G/C) is the number of G and C residues as a percentage oftotal residues in the duplex; (% F) is the percent formamide in solution(wt/vol); and L is the number of nucleotides in each strand of theduplex.

While not wishing to be bound by a single theory, the inventors notethat it is possible that certain modifications will result in modulatedresultant expression levels, including enhanced expression levels.Achievement of modulated resultant expression levels, preferablyenhanced expression levels, may be especially desirable in the case ofcertain, more aggressive forms of cancer, or when a more rapid and/oraggressive pattern of cell killing is warranted (due to animmunocompromised condition of the individual, for example).

Determination of TRE Activity

Activity of a TRE can be determined, for example, as follows. A TREpolynucleotide sequence or set of such sequences can be generated usingmethods known in the art, such as chemical synthesis, site-directedmutagenesis, PCR, and/or recombinant methods. The sequence(s) to betested can be inserted into a vector containing a promoter (if nopromoter element is present in the TRE) and an appropriate reporter geneencoding a reporter protein, including, but not limited to,chloramphenicol acetyl transferase (CAT), β-galactosidase (encoded bythe lacZ gene), luciferase (encoded by the luc gene), alkalinephosphatase (AP), green fluorescent protein (GFP), and horseradishperoxidase (HRP). Such vectors and assays are readily available, from,inter alia, commercial sources. Plasmids thus constructed aretransfected into a suitable host cell to test for expression of thereporter gene as controlled by the putative TRE using transfectionmethods known in the art, such as calcium phosphate precipitation,electroporation, liposomes, DEAE dextran-mediated transfer, particlebombardment or direct injection. TRE activity is measured by detectionand/or quantitation of reporter gene-derived mRNA and/or protein.Reporter protein product can be detected directly (e.g.,immunochemically) or through its enzymatic activity, if any, using anappropriate substrate. Generally, to determine cell specific activity ofa TRE, a TRE-reporter gene construct is introduced into a variety ofcell types. The amount of TRE activity is determined in each cell typeand compared to that of a reporter gene construct lacking the TRE. A TREis determined to be cell-specific if it is preferentially functional inone cell type, compared to a different type of cell.

Internal Ribosome Entry Site (IRES)

IRES elements were first discovered in picornavirus mRNAs (Jackson R J,Howell M T, Kaminski A (1990) Trends Biochem Sci 15(12):477-83) andJackson R J and Kaminski, A. (1995) RNA 1(10):985-1000). The presentinvention provides improved adenovirus vectors comprising co-transcribedfirst and second genes under transcriptional control of a heterologous,target cell-specific TRE, and wherein the second gene (i.e., codingregion) is under translational control of an internal ribosome entrysite (IRES). Any IRES may be used in the adenovirus vectors of theinvention, as long as they exhibit requisite function in the vectors.Example of IRES which can be used in the present invention include thoseprovided in Table I and referenced in Table II. Examples of IRESelements include the encephelomycarditis virus (EMCV) which iscommercially available from Novagen (Duke et al. (1992) J. Virol66(3):1602-9) the sequence for which is depicted in Table 1 (SEQ IDNO:1). Another example of an IRES element disclosed herein is the VEGFIRES (Huez et al. (1998) Mol Cell Biol 18(11):6178-90). This IRES has ashort segment and the sequence is depicted in Table 1 (SEQ ID NO:2).

The IRES promotes direct internal ribosome entry to the initiation codonof a downstream cistron, leading to cap-independent translation. Thus,the product of a downstream cistron can be expressed from a bicistronic(or multicistronic) mRNA, without requiring either cleavage of apolyprotein or generation of a monocistronic mRNA. Therefore, in oneillustrative embodiment of the present invention, an adenovirus vectorcomprising E1B under translational control of an IRES allows translationof E1B from a bicistronic E1A-E1B mRNA under control of a targetcell-specific TRE.

Internal ribosome entry sites are approximately 450 nucleotides inlength and are characterized by moderate conservation of primarysequence and strong conservation of secondary structure. The mostsignificant primary sequence feature of the IRES is a pyrimidine-richsite whose start is located approximately 25 nucleotides upstream of the3′ end of the IRES. See Jackson et al. (1990).

Three major classes of picornavirus IRES have been identified andcharacterized: (1) the cardio- and aphthovirus class (for example, theencephelomycarditis virus, Jang et al. (1990) Gene Dev 4:1560-1572); (2)the entero- and rhinovirus class (for example, polioviruses, Borman etal. (1994) EMBO J. 13:314903157); and (3) the hepatitis A virus (HAV)class, Glass et al. (1993) Virol 193:842-852). For the first twoclasses, two general principles apply. First, most of the 450-nucleotidesequence of the IRES functions to maintain particular secondary andtertiary structures conducive to ribosome binding and translationalinitiation. Second, the ribosome entry site is an AUG triplet located atthe 3′ end of the IRES, approximately 25 nucleotides downstream of aconserved oligopyrimidine tract. Translation initiation can occur eitherat the ribosome entry site (cardioviruses) or at the next downstream AUG(entero/rhinovirus class). Initiation occurs at both sites inaphthoviruses.

HCV and pestiviruses such as bovine viral diarrhea virus (BVDV) orclassical swine fever virus (CSFV) have 341 nt and 370 nt long 5′-UTRrespectively. These 5′-UTR fragments form similar RNA secondarystructures and can have moderately efficient IRES function(Tsukiyama-Kohara et al. (1992) J. Virol. 66:1476-1483; Frolov I et al.,(1998) RNA 4:1418-1435). Table I depicts the 5′-UTR region from HCVgenome sequence (GenBank accession D14853).

Leishmania RNA virus 1 (LRV1) is a double-stranded RNA virus. Its 128 ntlong 5′-UTR has IRES activity to facilitate the cap-independenttranslation, (Maga et al. (1995) Mol Cell Biol 15:4884-4889). Thisfragment also forms conserved stemloop secondary structure and at leastthe front part is essential.

Recent studies showed that both Friend-murine leukemia virus (MLV)5′-UTR and rat retrotransposon virus-like 30S (VL30) sequences containIRES structure of retroviral origin (Torrent et al. (1996) Hum Gene Ther7:603-612). These fragments are also functional as packing signal whenused in retroviruse derived vectors. Studies of avianreticuloendotheliosis virus type A (REV-A) show that its IRES mapsdownstream of the packaging/dimerization (E/DLS) sequence and theminimal IRES sequence appears to be within a 129 nt fragment (452-580)of the 5′ leader, immediately upstream of the gag AUG codon(Lopez-Lastra et al. (1997) Hum Gene Ther 8:1855-1865).

In eukaryotic cells, translation is normally initiated by the ribosomescanning from the capped mRNA 5′ end, under the control of initiationfactors. However, several cellular mRNAs have been found to be with IRESstructure to mediate the cap-independent translation (van der Velde, etal. (1999) Int J Biochem Cell Biol. 31:87-106). Examples areimmunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991)Nature 353:90-94), antennapedia mRNA of Drosophilan (Oh et al. (1992)Gene and Dev 6:1643-1653), fibroblast growth factor-2 (FGF-2) (Vagner etal. (1995) Mol Cell Biol 15:35-44), platelet-derived growth factor B(PDGF-B) (Bernstein et al. (1997) J Biol Chem 272:9356-9362),insulin-like growth factor II (Teerink et al. (1995) Biochim BiophysActa 1264:403408), and the translation initiation factor eIF4G (Gan etal. (1996) J Biol Chem 271:623-626). Table 1 depicts the 5′-noncodingregion for BiP and PDGF. Recently, vascular endothelial growth factor(VEGF) was also found to have IRES element (Stein et al. (1998) Mol CellBiol 18:3112-3119; Huez et al. (1998) Mol Cell Biol 18:6178-6190).

Apart from the oligopyrimidine tract, nucleotide sequence per se doesnot appear to be important for IRES function. Without wishing to bebound by theory, a possible explanation for the function of an IRES isthat it forms secondary and/or tertiary structures which orientparticular single-stranded regions of its sequence in athree-dimensional configuration that is conducive to interaction with amammalian ribosome (either ribosomal protein and/or ribosomal RNAcomponents) and/or initiation factor(s) and/or RNA binding proteinswhich interact with ribosomes and/or initiation factors. It is alsopossible that the three-dimensional structure of the IRES is determinedor stabilized by one or more RNA-binding proteins. Thus it is possibleto devise synthetic IRES sequences having similar single-strandedregions in a similar three-dimensional configuration.

In certain cases, one or more trans-acting cellular proteins may berequired for IRES function. For example, the HAV and entero/rhinovirusIRESes function inefficiently in vitro in reticulocyte lysates.Supplementation of a reticulocyte lysate with a cytoplasmic extract fromHeLa, Krebs II ascites, or L-cells restores activity ofentero/rhinovirus IRESes. See, for example, Brown et al. (1979) Virology97:396-405; and Dorner et al. (1984) J. Virol. 50:507-514. Activity ofthe HAV IRES in vitro is stimulated by liver cytoplasmic extracts. Glasset al. (1993) Virology 193:1047-1050. These observations indicate thatcell-specific translational regulation can be achieved through the useof a cell-specific IRES. Furthermore, coordinated cell-specifictranscriptional and translational regulatory elements can be included ina vector to further increase cell specificity of viral replication. Forexample, the combination of an AFP-TRE and a HAV-IRES can be used todirect preferential replication of a vector in hepatic cells. Thus, inone illustrative embodiment, a vector comprises an AFP-TRE regulatingthe transcription of a bicistronic E1A-E1B mRNA in which E1B translationis regulated by an ECMV IRES. In another illustrative embodiment, thevector comprises a probasin-TRE regulating the transcription of abicistronic E1A-E1B mRNA in which E1B translation is regulated by anECMV IRES. In yet another illustrative embodiment, a vector comprises aCMV-TRE regulating the transcription of a bicistronic E1A-E1B mRNA inwhich E1B translation is regulated by an ECMV IRES. In examplesdisclosed herein, E1B has a deletion of the 19-kDa region.

Examples of IRES which can be used in the present invention includethose provided in Table 12 and Table 13. In order to test for an IRESsequence which may be used in the present invention, a test vector isproduced having a reporter gene, such as luciferase, for example, placedunder translational control of an IRES to be tested. A desired cell typeis transfected with the vector containing the desired IRES-reporter geneand an assay is performed to detect the presence of the reporter gene.In one illustrative example, the test vector comprises a co-transcribedchloramphenicol transferase (CAT) and luciferase encoding genetranscriptionally driven by a CMV promoter wherein the luciferaseencoding gene is translationally driven by an IRES to be tested. Hostcells are transiently transfected with the test vector by means known tothose of skill in the art and assayed for the presence of luciferase.

IRES may be prepared using standard recombinant and synthetic methodsknown in the art, and as described in the Examples. For cloningconvenience, restriction sites may be engineered into the ends of theIRES fragments to be used.

Adenovirus Early Genes

The adenovirus vectors of the invention comprise adenovirus genes underthe control of a target cell-specific TRE. Preferably an adenovirus geneessential for replication. Any gene that is essential for adenovirusreplication, such as E1A, E1B, E2, E4 or any of the late genes, isuseful. The adenovirus may also comprise E3. In addition, one or more ofthe genes can be a transgene or heterologous gene. Any of the variousadenovirus serotypes can be used, such as, for example, Ad2, Ad5, Ad12and Ad40. For purposes of illustration, the Ad5 serotype is exemplifiedherein.

The E1A gene is expressed immediately (between 0 and 2 hours) afterviral infection, before any other viral genes. E1A protein is atrans-acting positive transcriptional regulatory factor, and is requiredfor the expression of the other early viral genes E1B, E2, E3, E4, andthe promoter-proximal major late genes. Despite the nomenclature, thepromoter proximal genes driven by the major late promoter are alsoexpressed during early times after Ad5 infection. Flint (1982) Biochem.Biophys. Acta 651:175-208; Flint (1986) Advances Virus Research31:169-228; and Grand (1987) Biochem. J. 241:25-38. In the absence of afunctional E1A gene, viral infection does not proceed, because the geneproducts necessary for viral DNA replication are not produced. Nevins(1989) Adv. Virus Res. 31:35-81. The transcription start site of Ad5 E1Ais at coordinate 498 and the ATG start site of the E1A protein is atcoordinate 560 in the virus genome.

The E1B protein is necessary in trans for transport of late mRNA fromthe nucleus to the cytoplasm. Defects in E1B expression result in poorexpression of late viral proteins and an inability to shut off host cellprotein synthesis. The promoter of E1B has been implicated as thedefining element of difference in the host range of Ad40 and Ad5:clinically Ad40 is an enterovirus, whereas Ad5 causes acuteconjunctivitis. Bailey et al. (1993) Virology 193:631; Bailey et al.(1994) Virology 202:695-706. The E1B promoter of Ad5 consists of asingle high-affinity recognition site for Sp1 and a TATA box, andextends from Ad5 nt 1636 to 1701.

Adenovirus E1B 19-kDa (19K) protein is a potent inhibitor of apoptosisand cooperates with E1A to produce oncogenic transformation of primarycells (Rao, et al., 1992, Cell Biology, 89:7742-7746). During productiveadenovirus infection, E1A stimulates host cell DNA synthesis, therebycausing cells to aberrantly go through the cell cycle. In response tocell cycle deregulation, the host cell undergoes apoptosis. As a defensemechanism, the E1B 19-kDa protein inhibits this E1A-induced apoptosisand allows assembly of viral progeny to be completed before the cellcommits suicide. E1B 19-kDa conducts anti-apoptotic function by multiplemechanisms. E1B 19-kDa inhibits the apoptosis of multiple stimuli,including E1a, p53 and TNF, for example. According to wild-type Ad5, theE1B 19-kDa region is located between nucleotide 1714 and nucleotide2244. The E1B 19-kDa region has been described in, for example, Rao etal., Proc. Natl. Acad. Sci. USA, 89:7742-7746.

In a preferred embodiment, expression of the E1A and E1B regions of theAd genome is facilitated in a cell-specific fashion by placing acell-specific TRE upstream of E1A and a internal ribosome entry sitebetween E1A and E1B.

The E2 region of adenovirus encodes proteins related to replication ofthe adenoviral genome, including the 72 kD DNA-binding protein, the 80kD precursor terminal protein and the viral DNA polymerase. The E2region of Ad5 is transcribed in a rightward orientation from twopromoters, termed E2 early and E2 late, mapping at 76.0 and 72.0 mapunits, respectively. While the E2 late promoter is transiently activeduring late stages of infection and is independent of the E1Atransactivator protein, the E2 early promoter is crucial during theearly phases of viral replication.

The E2 early promoter of Ad5 is located between nucleotides 27,050 and27,150, and consists of a major and a minor transcription initiationsite (the latter accounting for about 5% of E2 transcripts), twonon-canonical TATA boxes, two E2F transcription factor binding sites andan ATF transcription factor binding site. For a detailed review of E2promoter architecture see Swaminathan et al. (1995) Curr. Topics inMicro. and Imm. 199 part 3:177-194.

The E2 late promoter overlaps with the coding sequences of a geneencoded by the counterstrand and is therefore not amenable for geneticmanipulation. However, the E2 early promoter overlaps by only a few basepairs with sequences on the counterstrand which encode a 33 kD protein.Notably, an SpeI restriction site (Ad5 position 27,082) is part of thestop codon for the above mentioned 33 kD protein and convenientlyseparates the major E2 early transcription initiation site and TATA boxfrom the upstream E2F and ATF binding sites. Therefore, insertion of aheterologous TRE having SpeI ends into the SpeI site disrupts theendogenous E2 early promoter of Ad5 and allows TRE-regulated expressionof E2 transcripts.

An E3 region refers to the region of the adenoviral genome that encodesthe E3 products. The E3 region has been described in variouspublications, including, for example, Wold et al. (1995) Curr. TopicsMicrobiol. Immunol. 199:237-274. Generally, the E3 region is locatedbetween about 28583 and about 30470 of the adenoviral genome. An E3region for use in the present invention may be from any adenovirusserotype. An E3 sequence is a polynucleotide sequence that contains asequence from an E3 region. In some embodiments, the sequence encodesADP. In other embodiments, the sequence encodes other than ADP andexcludes a sequence encoding only ADP. As is well known in the art, theADP coding region is located in the E3 region within the adenoviralgenome from about 29468 bp to about 29773 bp; including the Y leader,the location of ADP is from about 28375 bp to about 29773 bp for Ad5.Other ADP regions for other serotypes are known in the art. An E3sequence includes, but is not limited to, deletions; insertions;fusions; and substitutions. An E3 sequence may also comprise an E3region or a portion of the E3 region. It is understood that, as an “E3sequence” is not limited to an “E3 region”, alternative referencesherein to an “E3 region” or “E3 sequence” do not indicate that theseterms are interchangeable. Assays for determining a functional E3sequence for purposes of this invention are described herein.

The E4 gene has a number of transcription products and encodes twopolypeptides (the products of open reading frames (ORFs) 3 and 6) whichare responsible for stimulating the replication of viral genomic DNA andstimulating late gene expression, through interaction with heterodimersof cellular transcription factors E2F-1 and DP-1. The ORF 6 proteinrequires interaction with the E1B 55 kD protein for activity while theORF 3 protein does not. In the absence of functional ORF 3- and ORF6-encoded proteins, efficiency of plaque formation is less than 10⁻⁶that of wild type virus.

To further increase cell-specificity of replication, it is possible totake advantage of the interaction between the E4 ORF 6 gene product andthe E1B 55 kD protein. For example, if E4 ORFs 1-3 are deleted, viralDNA replication and late gene synthesis becomes dependent on E4 ORF6protein. By generating such a deletion in a vector in which the E1Bregion is regulated by a cell-specific TRE, a virus is obtained in whichboth E1B and E4 functions are dependent on the cell-specific TRE whichregulates E1B.

Late genes relevant to the disclosed vectors are L1, L2 and L3, whichencode proteins of the virion. All of these genes (typically coding forstructural proteins) are probably required for adenoviral replication.All late genes are under the control of the major late promoter (MLP),which is located in Ad5 between nucleotides 5986 and 6048.

In one embodiment, an adenovirus early gene is under transcriptionalcontrol of a cell specific, heterologous TRE. In additional embodiments,the early gene is selected from the group including E1A, E1B, E2, E3,E4. In another embodiment, an adenovirus late gene is undertranscriptional control of a cell specific, heterologous TRE. In furtherembodiments, two or more early genes are under the control ofheterologous TREs that function in the same target cell. Theheterologous TREs can be the same or different, or one can be a variantof the other. In additional embodiments, two or more late genes areunder the control of heterologous TREs that function in the same targetcell. The heterologous TREs can be the same or different, or one can bea variant of the other. In yet another embodiment, one or more earlygene(s) and one or more late gene(s) are under transcriptional controlof the same or different heterologous TREs, wherein the TREs function inthe same target cell.

In some embodiments of the present invention, the adenovirus vectorcomprises the essential gene E1A and the E1A promoter is deleted. Inother embodiments, the adenovirus vector comprises the essential geneE1A and the E1A enhancer I is deleted. In yet other embodiments, the E1Apromoter is deleted and E1A enhancer I is deleted. In other embodiments,an internal ribosome entry site (IRES) is inserted upstream of E1B (sothat E1B is translationally linked), and a target cell-specific TRE isoperably linked to E1A. In still other embodiments, an (IRES) isinserted upstream of E1B (so that E1B is translationally linked), andtarget cell-specific TRE is operably linked to E1A, which may or may notmaintain the E1A promoter and/or enhancer I (i.e., the E1A promoterand/or enhancer I may be, but not necessarily be, deleted). In otherembodiments, the 19-kDa region of E1B is deleted. For adenovirus vectorscomprising a second gene under control of an IRES, it is preferred thatthe endogenous promoter of a gene under translational control of an IRESbe deleted so that the endogenous promoter does not interfere withtranscription of the second gene. It is preferred that the second genebe in frame with the IRES if the IRES contains an initiation codon. Ifan initiation codon, such as ATG, is present in the IRES, it ispreferred that the initiation codon of the second gene is removed andthat the IRES and second gene are in frame. Alternatively, if the IRESdoes not contain an initiation codon or if the initiation codon isremoved from the IRES, the initiation codon of the second gene is used.

Adenovirus Death Protein (ADP) Gene and Gene Product

In the construction of adenovirus vectors, the E3 region is oftendeleted to facilitate insertion of one or more TREs and/or transgenes.In some embodiments, however, the adenovirus death protein (ADP),encoded within the E3 region, is retained in an adenovirus vector. TheADP gene, under control of the major late promoter (MLP), appears tocode for a protein (ADP) that is important in expediting host celllysis. Tollefson et al. (1992) J. Virol. 66:3633; and Tollefson et al.(1996) J. Virol. 70:2296. Thus, inclusion of an ADP gene in a viralvector can render the vector more potent, making possible more effectivetreatment and/or a lower dosage requirement.

An ADP coding sequence is obtained preferably from Ad2 (since this isthe strain in which the ADP has been most fully characterized) usingtechniques known in the art, such as PCR. Preferably, the Y leader(which is an important sequence for correct expression of late genes) isalso obtained and placed in operative linkage to the ADP codingsequence. The ADP coding sequence (with or without the Y leader) is thenintroduced into an adenoviral genome, for example, in the E3 region,where expression of the ADP coding sequence will be driven by the MLP.The ADP coding sequence can, of course, also be inserted in otherlocations of the adenovirus genome, such as the E4 region.Alternatively, the ADP coding sequence can be operably linked to aheterologous TRE, including, but not limited to, another viral TRE or atarget cell-specific TRE (see infra). In another embodiment, the ADPgene is present in a viral genome such that it is transcribed as part ofa multi-cistronic mRNA in which its translation is associated with anIRES.

E3-Containing Target Cell-Specific Adenoviral Vectors

In some embodiments, the adenovirus vectors contain an E3 region, or aportion of an E3 region. Inclusion of the E3 region of adenovirus canenhance cytotoxicity of the target cell-specific adenoviral vectors ofthe present invention. Adenoviral vectors containing an E3 region maymaintain their high level of specificity and can be (a) significantlymore cytotoxic; (b) produce higher virus yield including extracellularvirus yield; (c) form larger plaques; (d) produce rapid cell death; and(e) kill tumors more efficiently in vivo than vectors lacking the E3region. The adenoviral vectors of this invention may contain the E3region or a portion of the E3 region. It is understood that, asinclusion of E3 confers observable and measurable functionality on theadenoviral vectors, for example, increased replication and production,functionally equivalent (in which functionality is essentiallymaintained, preserved, or even enhanced or diminished) variants of E3may be constructed. For example, portions of E3 may be used. A portionmay be, non-inclusively, either of the following: (a) deletion,preferably at the 3′ end; (b) inclusion of one or more various openreading frames of E3. Five proteins which are encoded by the Ad-E3region have been identified and characterized: (1) a 19-kDa glycoprotein(gp19k) is one of the most abundant adenovirus early proteins, and isknown to inhibit transport of the major histocompatibility complex classI molecules to the cell surface, thus impairing both peptide recognitionand clearance of Ad-infected cells by cytotoxic T lymphocytes (CTLs);(2) E3 14.7k protein and the E3 10.4k/14.5k complex of proteins inhibitthe cytotoxic and inflammatory responses mediated by tumor necrosisfactor (TNF); (3) E3 10.4k/14.5k protein complex down regulates theepidermal growth factor receptor, which may inhibit inflammation andactivate quiescent infected cells for efficient virus replication; (4)E3 11.6k protein (adenoviral death protein, ADP) from adenovirus 2 and 5appears to promote cell death and release of virus from infected cells.The functions of three E3-encoded proteins—3.6k, 6.7k and 12.5k—areunknown. A ninth protein having a molecular weight of 7.5 kDa has beenpostulated to exist, but has not been detected in cells infected withwild-type adenovirus. Wold et al. (1995) Curr. Topics Microbiol.Immunol. 199:237-274. The E3 region is schematically depicted in FIG. 6.These intact, portions, or variants of E3 may be readily constructedusing standard knowledge and techniques in the art. Preferably, anintact E3 region is used.

In the adenovirus vectors of the present invention, E3 may or may not beunder transcriptional control of native adenoviral transcriptionalcontrol element(s). The E3 promoter is located within the codingsequence for virion protein VIII, an essential protein which is highlyconserved among adenovirus serotypes. In some embodiments, E3 is undertranscriptional control of a heterologous TRE, including, but notlimited to, a target cell-specific TRE. Accordingly, in one embodiment,the invention provides an adenoviral vector, preferably replicationcompetent, that comprises E3 region (or a portion of E3) undertranscriptional control of a target cell-specific TRE. In otherembodiments, the E3 region is under transcriptional control of a nativeadenoviral TRE, and the vector further comprises an adenoviral geneessential for replication under transcriptional control of a targetcell-specific TRE. In other embodiments, the E3 region is undertranscriptional control of a target cell-specific TRE, and the vectorfurther comprises an adenoviral gene essential for replication undertranscriptional control of a target cell-specific TRE.

Transgenes Under Transcriptional Control of a Target Cell-Specific TRE

Various other replication-competent adenovirus vectors can be madeaccording to the present invention in which, in addition to having asingle or multiple adenovirus gene(s) under control of a targetcell-specific TRE, a transgene(s) is/are also under control of a targetcell-specific TRE and optionally under translational control of an IRES.Transgenes include, but are not limited to, therapeutic transgenes andreporter genes. Transgenes can be inserted into the adenoviral vector toproduce, for example, certain chemotherapeutic agents, chemoprotectants,chemosensitizers, radioprotectants and radiosensitizers. Examples ofsuch genes include, for example, genes encoding, p53, Adenovirus E1A,HSV-TK, Cytosine deaminase (CDA), Cytochrome p450, TAXOL™ or others.

Reporter Genes

For example, a target cell-specific TRE can be introduced into anadenovirus vector immediately upstream of and operably linked to anearly gene such as E1A or E1B, and this construct may further comprise asecond co-transcribed gene under translational control of an IRES. Thesecond gene may be a reporter gene. The reporter gene can encode areporter protein, including, but not limited to, chloramphenicol acetyltransferase (CAT), β-galactosidase (encoded by the lacZ gene),luciferase, alkaline phosphatase, a green fluorescent protein, and horseradish peroxidase. For detection of a putative cancer cell(s) in abiological sample, the biological sample may be treated with modifiedadenoviruses in which a reporter gene (e.g., luciferase) is undercontrol of a target cell-specific TRE. The target cell-specific TRE willbe transcriptionally active in cells that allow the target cell-specificTRE to function, and luciferase will be produced. This production willallow detection of target cells, including cancer cells in, for example,a human host or a biological sample. Alternatively, an adenovirus can beconstructed in which a gene encoding a product conditionally requiredfor survival (e.g., an antibiotic resistance marker) is undertranscriptional control of a target cell-specific TRE. When thisadenovirus is introduced into a biological sample, the target cells willbecome antibiotic resistant. An antibiotic can then be introduced intothe medium to kill the non-cancerous cells.

Therapeutic Transgenes

Transgenes also include genes which may confer a therapeutic effect,such as enhancing cytotoxicity so as to eliminate unwanted target cells.In this way, various genetic capabilities may be introduced into targetcells, particularly cancer cells. For example, in certain instances, itmay be desirable to enhance the degree and/or rate of cytotoxicactivity, due to, for example, the relatively refractory nature orparticular aggressiveness of the cancerous target cell. This could beaccomplished by coupling the target cell-specific cytotoxic activitywith cell-specific expression of, for example, HSV-tk and/or cytosinedeaminase (cd), which renders cells capable of metabolizing5-fluorocytosine (5-FC) to the chemotherapeutic agent 5-fluorouracil(5-FU). Using these types of transgenes may also confer a bystandereffect.

Other desirable transgenes that may be introduced via an adenovirusvector(s) include genes encoding cytotoxic proteins, such as the Achains of diphtheria toxin, ricin or abrin (Palmiter et al. (1987) Cell50: 435; Maxwell et al. (1987) Mol. Cell. Biol. 7: 1576; Behringer etal. (1988) Genes Dev. 2: 453; Messing et al. (1992) Neuron 8: 507;Piatak et al. (1988) J. Biol. Chem. 263: 4937; Lamb et al. (1985) Eur.J. Biochem. 148: 265; Frankel et al. (1989) Mol. Cell. Biol. 9: 415),genes encoding a factor capable of initiating apoptosis, sequencesencoding antisense transcripts or ribozymes, which among othercapabilities may be directed to mRNAs encoding proteins essential forproliferation, such as structural proteins, or transcription factors;viral or other pathogenic proteins, where the pathogen proliferatesintracellularly; genes that encode an engineered cytoplasmic variant ofa nuclease (e.g. RNase A) or protease (e.g. awsin, papain, proteinase K,carboxypeptidase, etc.), or encode the Fas gene, and the like. Othergenes of interest include cytokines, antigens, transmembrane proteins,and the like, such as IL-1, -2, -6, -12, GM-CSF, G-CSF, M-CSF, IFN-α,-β, -χ, TNF-α, -β, TGF-α, -β, NGF, and the like. The positive effectorgenes could be used in an earlier phase, followed by cytotoxic activitydue to replication.

Preparation of the Adenovirus Vectors

The adenovirus vectors of this invention can be prepared usingrecombinant techniques that are standard in the art. Generally, a targetcell-specific TRE is inserted 5′ to the adenoviral gene of interest,preferably an adenoviral replication gene, more preferably one or moreearly replication genes (although late gene(s) can be used). A targetcell-specific TRE can be prepared using oligonucleotide synthesis (ifthe sequence is known) or recombinant methods (such as PCR and/orrestriction enzymes). Convenient restriction sites, either in thenatural adeno-DNA sequence or introduced by methods such as PCR orsite-directed mutagenesis, provide an insertion site for a targetcell-specific TRE. Accordingly, convenient restriction sites forannealing (i.e., inserting) a target cell-specific TRE can be engineeredonto the 5′ and 3′ ends of a UP-TRE using standard recombinant methods,such as PCR.

Polynucleotides used for making adenoviral vectors of this invention maybe obtained using standard methods in the art, such as chemicalsynthesis, recombinant methods and/or obtained from biological sources.

Adenoviral vectors containing all replication-essential elements, withthe desired elements (e.g., E1A) under control of a target cell-specificTRE, are conveniently prepared by homologous recombination or in vitroligation of two plasmids, one providing the left-hand portion ofadenovirus and the other plasmid providing the right-hand region, one ormore of which contains at least one adenovirus gene under control of atarget cell-specific TRE. If homologous recombination is used, the twoplasmids should share at least about 500 bp of sequence overlap. Eachplasmid, as desired, may be independently manipulated, followed bycotransfection in a competent host, providing complementing genes asappropriate, or the appropriate transcription factors for initiation oftranscription from a target cell-specific TRE for propagation of theadenovirus. Plasmids are generally introduced into a suitable host cellsuch as 293 cells using appropriate means of transduction, such ascationic liposomes. Alternatively, in vitro ligation of the right andleft-hand portions of the adenovirus genome can also be used toconstruct recombinant adenovirus derivative containing all thereplication-essential portions of adenovirus genome. Berkner et al.(1983) Nucleic Acid Research 11: 6003-6020; Bridge et al. (1989) J.Virol. 63: 631-638.

For convenience, plasmids are available that provide the necessaryportions of adenovirus. Plasmid pXC.1 (McKinnon (1982) Gene 19:33-42)contains the wild-type left-hand end of Ad5. pBHG10 (Bett et al. (1994);Microbix Biosystems Inc., Toronto) provides the right-hand end of Ad5,with a deletion in E3. The deletion in E3 provides room in the virus toinsert a 3 kb target cell-specific TRE without deleting the endogenousenhancer/promoter. The gene for E3 is located on the opposite strandfrom E4 (r-strand). pBHG11 provides an even larger E3 deletion (anadditional 0.3 kb is deleted). Bett et al. (1994). Alternatively, theuse of pBHGE3 (Microbix Biosystems, Inc.) provides the right hand end ofAd5, with a full-length of E3.

For manipulation of the early genes, the transcription start site of Ad5E1A is at 498 and the ATG start site of the E1A coding segment is at 560in the virus genome. This region can be used for insertion of a targetcell-specific TRE. A restriction site may be introduced by employingpolymerase chain reaction (PCR), where the primer that is employed maybe limited to the Ad5 genome, or may involve a portion of the plasmidcarrying the Ad5 genomic DNA. For example, where pBR322 is used, theprimers may use the EcoRI site in the pBR322 backbone and the XbaI siteat nt 1339 of Ad5. By carrying out the PCR in two steps, whereoverlapping primers at the center of the region introduce a nucleotidesequence change resulting in a unique restriction site, one can providefor insertion of target cell-specific TRE at that site.

A similar strategy may also be used for insertion of a targetcell-specific TRE element to regulate E1B. The E1B promoter of Ad5consists of a single high-affinity recognition site for Sp1 and a TATAbox. This region extends from Ad5 nt 1636 to 1701. By insertion of atarget cell-specific TRE in this region, one can provide forcell-specific transcription of the E1B gene. By employing the left-handregion modified with the cell-specific response element regulating E1A,as the template for introducing a target cell-specific TRE to regulateE1B, the resulting adenovirus vector will be dependent upon thecell-specific transcription factors for expression of both E1A and E1B.In some embodiments, part or all of the 19-kDa region of E1B is deleted.

Similarly, a target cell-specific TRE can be inserted upstream of the E2gene to make its expression cell-specific. The E2 early promoter,mapping in Ad5 from 27050-27150, consists of a major and a minortranscription initiation site, the latter accounting for about 5% of theE2 transcripts, two non-canonical TATA boxes, two E2F transcriptionfactor binding sites and an ATF transcription factor binding site (for adetailed review of the E2 promoter architecture see Swaminathan et al.,Curr. Topics in Micro. and Immunol. (1995) 199(part 3):177-194.

The E2 late promoter overlaps with the coding sequences of a geneencoded by the counterstrand and is therefore not amenable for geneticmanipulation. However, the E2 early promoter overlaps only for a fewbase pairs with sequences coding for a 33 kD protein on thecounterstrand. Notably, the SpeI restriction site (Ad5 position 27082)is part of the stop codon for the above mentioned 33 kD protein andconveniently separates the major E2 early transcription initiation siteand TATA-binding protein site from the upstream transcription factorbinding sites E2F and ATF. Therefore, insertion of a targetcell-specific TRE having SpeI ends into the SpeI site in the 1-strandwould disrupt the endogenous E2 early promoter of Ad5 and should allowtarget cell-restricted expression of E2 transcripts.

For E4, one must use the right hand portion of the adenovirus genome.The E4 transcription start site is predominantly at about nt 35605, theTATA box at about nt 35631 and the first AUG/CUG of ORF I is at about nt35532. Virtanen et al. (1984) J. Virol. 51: 822-831. Using any of theabove strategies for the other genes, a UP-TRE may be introducedupstream from the transcription start site. For the construction offull-length adenovirus with a target cell-specific TRE inserted in theE4 region, the co-transfection and homologous recombination areperformed in W162 cells (Weinberg et. al. (1983) Proc. Natl. Acad. Sci.80:5383-5386) which provide E4 proteins in trans to complement defectsin synthesis of these proteins.

Adenoviral constructs containing an E3 region can be generated whereinhomologous recombination between an E3-containing adenoviral plasmid,for example, BHGE3 (Microbix Biosystems Inc., Toronto) and anon-E3-containing adenoviral plasmid, is carried out.

Alternatively, an adenoviral vector comprising an E3 region can beintroduced into cells, for example 293 cells, along with an adenoviralconstruct or an adenoviral plasmid construct, where they can undergohomologous recombination to yield adenovirus containing an E3 region. Inthis case, the E3-containing adenoviral vector and the adenoviralconstruct or plasmid construct contain complementary regions ofadenovirus, for example, one contains the left-hand and the othercontains the right-hand region, with sufficient sequence overlap as toallow homologous recombination.

Alternatively, an E3-containing adenoviral vector of the invention canbe constructed using other conventional methods including standardrecombinant methods (e.g., using restriction nucleases and/or PCR),chemical synthesis, or a combination of any of these. Further, deletionsof portions of the E3 region can be created using standard techniques ofmolecular biology.

Insertion of an IRES into a vector is accomplished by methods andtechniques that are known in the art and described herein supra,including but not limited to, restriction enzyme digestion, ligation,and PCR. A DNA copy of an IRES can be obtained by chemical synthesis, orby making a cDNA copy of, for example, a picornavirus IRES. See, forexample, Duke et al. (1995) J. Vvirol. 66(3):1602-9) for a descriptionof the EMCV IRES and Huez et al. (1998), Mol. Cell. Biol.18(11):6178-90) for a description of the VEGF IRES. The internaltranslation initiation sequence is inserted into a vector genome at asite such that it lies upstream of a 5′-distal coding region in amulticistronic mRNA. For example, in a preferred embodiment of anadenovirus vector in which production of a bicistronic E1A-E1B mRNA isunder the control of a target cell-specific TRE, the E1B promoter isdeleted or inactivated, and an IRES sequence is placed between E1A andE1B. In other embodiments, part or all of the 19-kDa region of E1B isdeleted. IRES sequences of cardioviruses and certain aphthovirusescontain an AUG codon at the 3′ end of the IRES that serves as both aribosome entry site and as a translation initiation site. Accordingly,this type of IRES is introduced into a vector so as to replace thetranslation initiation codon of the protein whose translation itregulates. However, in an IRES of the entero/rhinovirus class, the AUGat the 3′ end of the IRES is used for ribosome entry only, andtranslation is initiated at the next downstream AUG codon. Accordingly,if an entero/rhinovirus IRES is used in a vector for translationalregulation of a downstream coding region, the AUG (or other translationinitiation codon) of the downstream gene is retained in the vectorconstruct.

Methods of packaging polynucleotides into adenovirus particles are knownin the art and are also described in co-owned PCT PCT/US98/04080.

The following examples are offered by way of illustration and should notbe considered as limiting the scope of the invention. The specificexamples exemplify the adenovirus 5 serotype, however, persons skilledin the art will realize these techniques may be applied to otheradenoviral serotypes.

EXAMPLES

Table 4 summarizes descriptions of the various replication-competenttarget-cell specific adenoviral constructs used in these studies, anddescribed previously herein. Preparation of these adenoviral vectors(including their components) employ standard techniques in the art. Seealso PCT/US99/03117, PCT/US98/16312, PCT/US98/04133, PCT/US98/04132,PCT/US98/04084, PCT/US98/04080, PCT/US97/13888, PCT/US96/10838,PCT/US95/00845. In these publications, a CV designation is also denotedas CN. For example, CV706 is also denoted as CN706. TABLE 4 SummaryDescription of Adenoviral Constructs ADENOVIRAL TARGET E3 E1A E1B VECTORCELL TYPE E1A TRE E1B TRE +/− PROMOTER PROMOTER CV706 Prostate PSE N/A− + + CV787 Prostate PB PSE + + + CV790 Liver AFP AFP + + + (0.827 kb)(0.827 kb) CV829 Bladder hUPII mUPII + − + CV859 Melanoma tyrosinaseIRES + − − CV873 Colorectal CEA IRES + − − Breast CV874 Bladder mUPII(2kb) IRES + − − CV875 Bladder hUPII(1 kb) IRES + − − CV876 BladderhUPII(2 kb) IRES + − − CV877 Bladder mUPII(1 kb) hUPII(1 kb) + − − CV890Liver AFP IRES + − − CV884 Bladder hUPII IRES + − − (1.8 kb)For all constructs the E1A enhancer is present.PSA, prostate specific enhancer/promoter;PB, rat probasin promoter;AFP, α-fetoprotein promoter;mUPII, mouse uroplakin II promoter;hUPII, human uroplakin II promoter;tyrosinase, melanocyte specific TRE;IRES, internal ribosome entery site.

Example 1 Treatment of In Vitro Tumor Cells with Combined Prostate CellSpecific Adenoviral Vector CV787 and Chemotherapy and In Vivo Assessment

In Vitro Assessment.

CV787 is a prostate-specific, replication competent adenovirus vectorthat preferentially replicates in prostate cancer cells. In this vector,E1A is under transcriptional control of a 452 bp PB TRE, and E1B isunder transcriptional control of 1.6 kb PSA-TRE. CV787 alone can, in asingle intratumoral dose (1×10⁸ particles per mm³ of tumor) or a singleintravenous dose (1×10¹¹ particles per animal) eliminate establishedtumors within 6 weeks in nude mouse xenografts. The data belowdemonstrate that CV787-mediated, replication-dependent oncolyticcytotoxicity can be enhanced in conjunction with standardchemotherapeutic agents including paclitaxel (TAXOL™), doxorubicin,mitoxantrone and docetaxel (TAXOTERE™), while the specificity ofCV787-based cytopathogenicity remains specific to prostate cancer cells.These data suggest that the combination of CV787 with chemotherapy ismore effective than chemotherapy treatment alone or virus treatmentalone.

Cell Lines and Culture

The human LNCaP (prostate carcinoma), HBL-100 (breast epithelia), andOVCAR-3 (ovarian carcinoma) were obtained from the American Type CultureCollection (Rockville, Md.). The human embryonic kidney cell line, 293,which expresses the adenoviral E1A and E1B gene products serves as aproduction cell line, and was purchased from Microbix: Biosystem, Inc.(Toronto, Canada). Cells were maintained at 37 C with 5% CO₂ in RPMI1640 (Life Technologies, Gaithersburg, Md.) supplemented with 100units/ml penicillin and 100 μg/ml of streptomycin (Life Technologies,Gaithersburg, Md.).

Chemotherapeutic Agents and Virus

Paclitaxel (TAXOL™, Bristol-Myers Squibb, Princeton, N.J.), docetaxel(TAXOTERE™, Rhone-Poulenc Rorer Pharmaceuticals, Inc., Collegeville,Pa.), and the chemotherapeutic agents listed in Table 5, were purchasedfrom the Stanford University Hospital pharmacy (Palo Alto, Calif.).These agents were diluted with medium without fetal bovine serum (FBS)just before use for in vitro studies and with 0.9% NaCl for in vivostudies.

CV787 is a prostate-specific replication-competent adenovirus. Yu et al.(1999) Cancer Res. 59:4200. Two prostate-specific transcription responseelements (TRE), the rat probasin promoter and the humanprostate-specific antigen (PSE) promoter/enhancer, were insertedupstream of the E1A and E1B encoding regions in the viral genome,respectively, using methods known in the art. The expression of the E1Agene and the E1B gene are then controlled by these TREs.

Combination Study of CV787 with Paclitaxel (TAXOL™), Docetaxel(TAXOTERE™) or Other Chemotherapeutic Agents In Vitro

In preliminary experiments, we examined the chemosensitivity todifferent agents, as well as the oncolytic effect of CV787 in theprostate carcinoma LNCaP cells. Cells were plated in 96-well plates at adensity of 20,000 cells per well. Twenty-four hours later, the cellswere infected with CV787 at various multiplicities of infection (MOI).Subsequently, medium (50 μl) containing 10% heat-inactivated serum andvarious concentrations of chemotherapeutic agents were added to theappropriate wells. Cells were incubated at 37° C. in 5% CO₂ for anadditional two days. Cell viability was measured using the MTT assay.Mosmann et al., (1983). Briefly, 50 μl of 1 mg/ml MTT vital dye (Sigma,St. Louis, Mo.) was added to each well and allowed to incubate for 3 hat 37° C. and 5% CO₂. Then, plates were drained to remove untransformedMTT and blot. 100 μl of isopropanol was added to each well, the platewas incubated for 15 minutes and vigorously shaken (Microshaker II,Dynatech) in order to ensure solubilization of the blue formazan. Theoptical density of each well was quantitated using an automatic platereader (Molecular Devices, Sunnyvale, Calif.) with a 560 nm testwavelength and 690 nm reference wavelength. Cell viability was definedas the ratio of the mean absorbance of 9 treatment wells minus the blankto the mean absorbance of 6 untreated matched controls minus the blank.Blank is defined as the mean absorbance of six wells containing mediumalone. Each experiment was performed at least twice.

Other chemotherapeutics were tested, using the protocols describedabove.

Results of In Vitro Experiments

To study potential synergy or enhancement in treatment whenadministering CV787 and chemotherapy in vitro, the effectiveness of thecombined treatment at several concentrations of paclitaxel, ranging from0-62.5 nM, or docetaxel, ranging from 125-250 nM, with CV787 at variousMOIs, ranging from 0-10 MOI, was evaluated in the prostate carcinomaLNCaP cells. Cells were treated with CV787 and paclitaxel or docetaxeland the cell viability was determined at various time points aftertreatment by an MTT assay, as shown in FIGS. 2-4. FIG. 2 presents datafor treatment with a combination of CV787 (MOI 0.01) and paclitaxel(6.25 nM), showing the synergistic cytotoxicity of the combinationtreatment compared to virus alone or chemotherapy alone. An enhancedcytotoxicity was observed in the combination treatment between CV787 andpaclitaxel. For example, CV787 at an MOI of 0.01 produced 85% cellsurvival 6 days after virus infection and paclitaxel at 6.25 nM showed100% survival in LNCaP. When CV787 and paclitaxel were combined at theseconcentrations, cell survival dropped to 18%, demonstrating a greatereffect than just an additive effect. To determine whether the timing ofadministration for the testing articles affected the combined oncolyticeffect, LNCaP cells were treated with paclitaxel for 24 hours before orafter infection with CV787. Results showed that there were nosignificant differences in oncolytic activity between cells treated withpaclitaxel before or after infection with CV787.

Cytotoxicity was also measured for the combination treatment of CV787and docetaxel, FIGS. 3 and 4, and synergistic effects were observed.LNCaP cells were infected with CV787 at an MOI of 0.01 after a 24 hourincubation with docetaxel at 3.12 nM and cell viability was determinedby MTT, as shown in FIG. 3. The cell survival was 25% of the control atday 7 post treatment, whereas CV787 alone produced 95% cell survival anddocetaxel alone showed 95% cell survival in prostate carcinoma LNCaPcells. No significant difference in the effectiveness of the combinedtherapy of docetaxel and CV787 infection was observed by varying thetime of virus administration. As presented in FIG. 3, LNCaP cellstreated with docetaxel for 24 hours, then infected with CV787 producedsimilar cell viability to the treatment of which the LNCaP cells wereinfected with CV787 24 hours prior to docetaxel FIG. 4.

The protocols described above were used to screen a number of differentchemotherapeutic agents from various classes of chemotherapeutics. Theresults are presented in FIGS. 5-9 and are summarized in Table 5, below.The summarized results are for experiments in which drug was added 24hours before the introduction of the virus, except in the case ofdoxorubicin, in which the virus was added 24 hours prior to theadministration of the drug. FIGS. 3-4 compare the order ofadministration for a combination of docetaxel and CV787. CV787 wasadministered at MOI of either 0.1 or 0.01 as indicated in FIGS. 5-9.Chemotherapeutics were administered in the following amounts: paclitaxel(6.25 nM); docetaxel (3.12 nM); mitoxantrone (100 nM); etoposide (500ng/ml); doxorubicin (50 ng/ml); cisplatin (8.25 μM); 5-fluorouracil (35μM); estramustine (5 mg/ml); gemcitabine (50 ng/ml); flutamide (15ng/ml); goserelin (50 μg/1 g); leuprolide (5 nM); and vinblastine (80mg/ml). TABLE 5 Synergistic Effects of CV787/ChemotherapeuticCombinations TARGET/ CHEMOTHERAPEUTIC VIRUS CELL LINE AGENT CLASS OFAGENT SYNERGY CV787 Prostate cancer/ 5-Fluorouracil Antimetabolites(acting as Yes LNCaP (5-FU) pseudosubstrate for essential enzymaticreactions) CV787 Prostate cancer/ Cisplatin Alkylating agent (Plantinum-Yes LNCaP containing agents - Causing single- and double-strand break inDNA) CV787 Prostate cancer/ Doxorubicin Antibiotics (anticycline; YesLNCaP interrupting DNA replication and transcription, causing strandbreak) CV787 Prostate cancer/ Estramustine Alkylating agent Yes LNCaPCV787 LNCaP Etoposide Plant alkaloid (inhibiting the Yes assembly ofmicrotubules and disrupting mitosis CV787 Prostate cancer/ MitoxantroneAntibiotics (anticycline) Yes LNCaP CV787 Prostate cancer/ TAXOTERE ™Plant alkaloids Yes LNCaP (docetaxel) CV787 Prostate cancer/ TAXOL ™Plant alkaloids Yes LNCaP (paclitaxel) CV787 Prostate cancer/Gemcitabine Antimetabolite No LNCaP CV787 Prostate cancer/ FlutamideAnti-androgen No LNCaP CV787 Prostate cancer/ ZOLADEX ™ Hormonal analogNo LNCaP (goserelin) CV787 Prostate cancer/ LUPRON ™ Testosterone analogNo LNCaP (leuprolide) CV787 Prostate cancer/ Vinblastine Plant alkaloidsNo LNCaP

The following experiments were designed to test the specificity andviability of the replication-competent target cell-specific adenoviralvectors described herein in the presence of antineoplastic(chemotherapeutic) agents.

Virus Yield

Virus yield was determined to characterize the specificity ofcombination treatment of CV787 and paclitaxel or docetaxel. 5×10⁵ 293,LNCaP, HBL-100 and OVCAR-3 cells were plated in duplicate into six-wellplates. Twenty-four hours later, medium was aspirated and replaced with1.0 ml of serum-free RPMI 1640 containing CV787 at a MOI of 1 PFU(plaque-forming unit) per cell. After a 4 hour incubation at 37° C. with5% CO₂, cells were washed twice with pre-warmed phosphate bufferedsaline (PBS), and 2 ml of complete RPMI 1640 containing the indicatedchemotherapeutic agents were added into each well in concentrations andamounts as indicated below. After an additional 72 hours, the cells werescraped into the culture medium, and the cells were lysed by threefreeze-thaw cycles. The supernatant of each duplicate point was testedfor virus production by triplicate plaque assay for 12 days undersemisolid agarose on 293 cells. Yu et al. Cancer Research (1999)59:1698.

Paclitaxel Does Not Inhibit CV787 Replication

Paclitaxel (TAXOL™) and docetaxel (TAXOTERE™) are antineoplastic agentsbelonging to the taxoid family. They are novel antimicrotubule agentsthat promote the assembly of microtubules from tubulin dimers andstabilize microtubules; by preventing depolymerization. This stabilityresults in the inhibition of the normal dynamic reorganization of themicrotubule network that is essential for vital interphase and mitoticcellular functions. In addition, they induce abnormal arrays or“bundles” of microtubules throughout the cell cycle and multiple astersof microtubules during mitosis.

To examine the effect of paclitaxel on the virus replication, we ran avirus yield assay. LNCaP cells were infected with CV787 at a MOI of 0.1for 4 hours, followed by incubation in RPMI 1640 containing paclitaxelat a final concentration of 6.25 nM. Cells were harvested 6 dayspost-infection and the number of infectious virus particles weredetermined on 293 cells by a standard plaque assay. As shown in theFigures, cells treated with CV787 and paclitaxel produced 7,000 pfu percell, while the cells infected with CV787 alone generated about 4,600pfu per cell, suggesting that paclitaxel does not inhibit CV787replication.

In addition, the chemotherapeutics mitoxantrone, doxorubicin andetoposide were also tested with CV787 according to the above protocol.None of these chemotherapeutics, from different classes of agents,showed a reduction in viral yield compared to CV787 withoutchemotherapeutic agent.

Paclitaxel Does Not Alter CV787's Specificity

In order to evaluate whether addition of paclitaxel could change thespecificity of CV787's oncolytic activity, we tested viral replicationefficiency in four cell lines including a permissive cell line LNCaP,and two non-permissive cell lines, HBL-100 (breast epithelia) andOVCAR-3 (ovarian carcinoma). These three cell lines were infected witheither CV787 at an MOI of 0.1 or CV787 and paclitaxel, with a finalpaclitaxel concentration of 6.25 nM in the medium. Progeny virus yieldwas determined 48 hours after infection by plaque assay on 293 cells.Results presented in FIG. 12 show that prostate cancer (LNCaP) treatedwith CV787 and paclitaxel produced a similar burst size to the cellsinfected with CV787 alone, which produced about 800 pfu per cell. CV787replicated poorly in the non-prostate cancer cells tested (HBL-100 andOVCAR-3), producing 1000 to 10,000-fold lower virus yield compared tothe burst size in LNCaP cells. Interestingly, the burst size in theLNCaP cells treated with CV787 and paclitaxel is similar to that in thecells infected by CV787 alone. These data indicate that CV787 in thepresence of paclitaxel replicates efficiently in prostate cancer cells,but is still attenuated in non-prostate cancer cells. Combinationtreatment does not change CV787 replication efficiency in thenon-prostate cells and retains a high selectivity. Similar results wereobtained for combinations of CV787 and mitoxantrone (MXT) anddoxorubicin (DOXO).

To further assess the specificity of the combination treatment of CV787and paclitaxel, the viability of various infected cells was estimatedusing the MTT assay to measure mitochondrial activity. HEK-293, LNCaP,HBL-100 and OVCAR-3 cells were infected with CV787 at an MOI of 0.1 inthe presence or absence of paclitaxel. The percentage of cell viabilityin the combination treatment group versus paclitaxel treatment group wasplotted in FIG. 13. Combination of CV787 and paclitaxel was toxic to293, a permissive production cell line, and LNCaP cells, prostate cancercells, but not to HBL-100, normal breast epithelial cells, and OVCAR-3cells, ovarian cancer cells. There were no surviving LNCaP cells 9 daysafter infection. In contrast, the viability of HBL-100 and OVCAR-3 cellstreated with CV787 and paclitaxel was similar to that of cells treatedwith paclitaxel (ratio of cell survival between combination group andpaclitaxel group was approximately 1). The results suggest that thepresence of paclitaxel does not alter the cytotoxic effect of CV787.

Similar results were observed using the above protocols and acombination of CV787 and mitoxantrone FIG. 14.

In Vivo Assesment

Using the PSA+ LNCaP xenograft model of prostate cancer, a single i.v.dose of 1×10⁸ particles CV787 and docetaxel in combination eliminateslarge pre-existent distant tumors. Toxicity studies do not show asynergistic increase of toxicity of CV787 and taxane. These experimentsdemonstrate a synergistic antitumor efficacy for CV787 when combinedwith taxane, and demonstrate an in vivo single-does curative therapeuticindex for CV787 of over 1000:1.

Cell Viability

MTT assays were performed by seeding LNCaP, HBL-100, OVCAR-3, HepG2, and293 cells at 5000 cells per well in a 96 well plate (Falcon) 48 hr priorto infection as previously described (Denizot, 2000, J Immunol. Methods89:271-7.) with modifications. Cells were either infected with CV787 atan MOI of 2 PFU/cell or treated with the indicated chemotherapeuticagents (Paclitaxel at 6.25 nM and Docetaxel at 3.12 nM). Cell viabilitywas measured at the times indicated by removing the media and replacingit with 50 μl of a 1 mg/ml solution of MTT (3-(4,5-Dimethylthiazol-2-yly2,5-diphenyl-2H-tetrazolium bromide) (Sigma, St. Louis, Mo.) andincubating for 3 hrs at 3 hrs at 37° C. After removing the MTT solution,the crystals remaining in the wells were solubilized by the addition of50 μl of isopropanol followed by vigorous shaking. The absorbency wasdetermined using a microplate reader (Molecular Dynamics) at 560 nm(test wavelength) and 690 nm (reference wavelength). The percentage ofsurviving cells was estimated by dividing the OD₅₅₀-OD₆₅₀ of virusinfected cells by the OD₅₅₀-OD₆₅₀ of mock infected cells. 12 replicasamples were taken for each time point and each experiment was repeatedat least three times.

Statistical Analysis

The dose-response interactions between taxane and CV787 at the point ofIC₅₀ were evaluated by the isobologram method of Steel and Peckham(Steel, 1993, Int. J. Rad. One. Biol. Phys. 5:85.) as modified by Aoe etal. (Aoe, K. et al. 1999, Anticancer Res. 19:291-299.) The IC₅₀ wasdefined as the concentration of drug that produced 50% cell growthinhibition, i.e. 50% reduction in absorbance. Cells were exposed todrugs sequentially for 24 h and cell viability was determined by the MTTassay after 6 days. The dose-response curves were plotted withCurveExpert (Version 1.34) on a semilog scale as a percentage of thecontrol, the absorbance of which was obtained from the samples notexposed to the drugs. IC₅₀ value of CV787 and taxane in LNCaP was thendetermined. Based upon the dose-response curves of CV787 alone andtaxane alone, isobolograms (three isoeffect curves, model 1 and model 2lines) were computed. The envelope of additivity, surrounded by model 1and model 2 isobologram lines, was constructed from the dose responsecurves of CV787 alone and taxane alone. The observed data were comparedwith the predicted maximum and minimum data for presence of synergism,additivity, or antagonism by a statistical analysis using the Stat View4.01 software program (Abacus Concepts, Berkeley, Calif.). When the datapoints of the drug combination fall within the area surrounded by model1 and/or model 2 lines (i.e. within the envelope of additivity), thecombination is described as additive. A combination that gives datapoints to the left of the envelope of additivity can be described assupraadditive (synergism) and a combination that gives data points tothe right of the envelope of additivity, can be described as subadditive(antagonistic) (Kano, Y. et al. 1998, Cancer Chemo. Pharm. 42:91-98.)Fractional tumor volume (FTV) relative to untreated controls wasdetermined as described previously (Yokoyama, Y. et al., 2000, CancerRes. 60:2190-2196.).

One-Step Growth Curve and Virus Yield

One-step growth curves of CV787 in the presence or absence of docetaxelwere performed in LNCaP cells to determine burst size. Monalayers ofLNCaP cells were infected at a multiplicity of 2 PFU/cell with CV787.After a 4 hour incubation at 37° C. with 5% CO₂, cells were washed twicewith pre-warmed PBS, and 2 ml of complete RPMI 1640 containing docetaxelat a concentration of either 0 nM or 3.12 nM was added into each well.At the indicated times thereafter, duplicate cell samples were harvestedand lysed by three cycles of freeze-thawing. Virus was titered intriplicate (Yu, D.-C. et al., 1999, Cancer Res. 59:1498-1504.).

Virus yield was used to determine if CV787 retained specificity in thecombination treatment of CV787 and taxane. 5×10⁵ cells of 293, LNCaP,HBL-100, HepG2 and OVCAR-3 were plated in duplicate into six-wellplates. Twenty-four hours later, medium was aspirated and replaced with1.0 ml of serum-free RPMI 1640 containing CV787 at an MOI of 1 PFU(plaque-forming unit) per cell. After a 4 hour incubation at 37° C. with5% CO₂, cells were washed twice with pre-warmed PBS, and 2 ml ofcomplete RPMI 1640 containing the indicated taxane was added to eachwell. After an additional 72 hours, cells were scraped into the culturemedium, and lysed by three freeze-thaw cycles. Virus production wasmonitored by triplicate plaque assay (Yu, D.-C., et al., 1999, CancerRes. 59:1498-1504.).

Immunoblots

LNCaP cells treated with CV787, taxane, or both CV787 and taxane, wereincubated for the indicated times. Cells were washed with cold PBS, andlysed for 30 min on ice in 50 mM Tris, pH8.0, 150 mM NaCl, 1% IGEPALCA360 (NP40 equivalent from Sigma), 0.5% sodium deoxycholate, andprotease inhibitor cocktail (Roche, Palo Alto, Calif.). After 30 mincentrifugation at 4° C., the supernatant was removed and proteinconcentration was determined by the ESL protein assay kit (Roche). Fiftymicrograms of protein/lane were separated on 8-16% SDS-PAGE andelectroblotted onto Hybond ECL membranes (Amersham Pharmacia,Buckinghamshire, England). The membrane were blocked overnight in PBST(PBS with 0.1% Tween-20) supplemented with 5% nonfat dry milk. Primaryantibody incubation was done at room temperature for 2-3 hrs withPBST/1% nonfat dry milk diluted antibody, followed by wash and 1 hrincubation with diluted horseradish peroxidase-conjugated secondaryantibody. Enhanced chemiluminescence (ECL; Amersham Pharmacia) was usedfor detection. Antibodies for p53 and poly-ADP-ribose-polymerase werefrom Roche. Antibodies against Fas/Fas-L, caspase 7, Bcl-2, Bcl-XL, Baxand secondary antibodies were purchased from Santa Cruz BiotechnologyInc. (Santa Cruz, Calif.). All antibodies were used accordingmanufacturer's instruction. For quantifying the bands, the gels werescanned and bands were analyzed by Multi-Analyst software (Blo-Rad).

In Vivo Antitumor Efficacy

Six to eight week old athymic Balb/c nu/nu mice were obtained fromSimonson Laboratories (Gilroy, Calif.) and acclimatized to laboratoryconditions one week prior to tumor implantation. Xenografts wereestablished by injecting 1×10⁶ LNCaP cells, suspended in 100 pd of RPMI1640 and 100 μd of matrigel, subcutaneously near the small of the back.When tumors reached between 400 mm³ and 600 mm³, mice were randomizedinto groups of four. The first group received 1×10¹⁰ particles of CV787at day 1 via the tail vein intravenously (i.v.). CV787 was diluted in0.1 ml lyophilized buffer (5% sucrose, 1% glycine, 1 mM MgCl₂, 0.05%Tween-80 in 10 mM Tris buffer) and injected into the tail vein using a28-gauge needle. The second group was given taxane only. Paclitaxel wasintraperitoneally administered at a dose of 20 mg/kg, daily for 4 daysstarting at day 2. Docetaxel was intravenously administered at a dose of5 or 12.5 mg/kg at day 2, 5 and 8. The third group was given CV787(i.v.) at day 1 and taxane at the same doses and schedule as the secondgroup. As a control, a fourth group was treated with 0.1 ml of normalsaline (i.e. control) i.v. at day 1 and then i.p. or i.v. for 4 days.The dose and route of administration of paclitaxel were selectedaccording to studies in nude mice (Riondel, J. et al., 1986, CancerChemother Pharmacol. 17:137-42.) (Chahinian, A. P. et al., 1998, J.Surg. One. 67:104-111.). For docetaxel, the dose was selected based onthe human clinical dose. (RPR Pharm. Inc., Collegeville, Pa.) anddetermined by a dose-range finding study in nude mice. Tumors weremeasured weekly in two dimensions by external caliper and volume wasestimated by the formula [length (mm)×width (mm)^(2])/2 (7). Animalswere humanely killed when their tumor burden became excessive. Serum washarvested weekly by retro-orbital bleed. The difference in mean tumorvolume between treatment groups was compared for statisticalsignificance using the unpaired, two-tailed, t-test. Blood samples werecollected at various time points for determining prostate-specificantigen. Federal and institutional guidelines for animal care werefollowed.

Immunohistochemistry

Four groups of mice (n=6) were treated with vehicle, CV787 (1×10¹⁰particles per animal), paclitaxel (15 mg/kg) or a combination of CV787and paclitaxel at these identical doses. Half the animals weresacrificed on day 9 and the other half on day 16. Tumors were fixed in10% neutral buffered formalin, embedded in paraffin and sectioned usingstandard procedures. For detecting adenovirus, tissue sections wereblocked with ready-to-use normal rabbit serum (Biogenex, San Carlos,Calif.) for 20 min and incubated with goat anti-Ad antibody (BiodesignInternational, Kennebunkport, Me.) diluted 1:200 in PBS for 30 min.Alkaline phosphatase staining was performed using Super Sensitive™streptaviden-blotin alkaline phosphatase reagents and Fast Red™chromogen (Biogenex) as suggested by the manufacturer. Sections werecounterstained with Gill's hematoxylin and mounted with Gel Mount™(Biomedia, Foster City, Calif.).

Apoptotic cells were detected using M30 monoclonal antibody withreagents from the M30 CytoDEATH™ kit (Roche Molecular Biochemicals,Indianapolis, Ind.) as suggested by the manufacturer. Paraffin-embeddedtumor sections were heated in citric acid buffer for 15 min to retrieveantigen, hybridized with M30 antibody, then counterstained with Harrieshematoxilin (Roche Molecular Biochemicals). The stained sections wereanalyzed under a light microscope and pictures of representativesections taken.

Isobolograms were also generated to show the synergy between CV787 anddocetaxel. Dose-response curve analysis indicated that the IC₅₀ at day 5in LNCaP cells for CV787 and docetaxel was 0.368 MOI and 8.14 nM,respectively. The combined data points fell to the left of the envelopeof additivity, or restated the IC50 in LNCaP cells of CV787 incombination with docetaxel occurred at smaller doses than that predictedfrom the use of CV787 or docetaxel alone. Thus, sequential exposure toCV787 followed by docetaxel produced synergistic effects.

To determine whether the timing of administration for the testedcompounds affected the combined cytotoxic effect, LNCaP cells weretreated with paclitaxel for 24 hours before or after infection withCV787. There were no significant differences in cytotoxic activitybetween cells treated with paclitaxel before infection, after infection,or simultaneously with CV787. Similar results were obtained fordocetaxel.

Taxane Increases CV787 Burst Size in LNCaP Calls

Paclitaxel and docetaxel are antineoplastic agents belonging to thetaxane family. They are novel antimicrotubule agents that promote theassembly of microtubulas from tubulin dimers and stabilize microtubulesby preventing depolymerization. This stability results in the inhibitionof the normal dynamic reorganization of the microtubule network that isessential for vital interphase and mitotic cellular functions(Blagosklonny, M. V. et al., 2000, J. Urol. 163:1022-6.). In addition,the taxanes induce abnormal arrays or “bundles” of microtubulesthroughout the cell cycle and multiple asters of microtubules duringmitosis. One possible explanation for the synergy seen with taxane andCV787 is that taxane may augment the ability of CV787 to replicate inLNCaP cells.

To examine the effect of paclitaxel and docetaxel on virus replication,we performed the one-step growth curve. LNCaP cells were infected withCV787 at an MOI of 1 for 4 hrs, followed by incubation in RPMI 1640containing docetaxel at a final concentration of 3.12 nM. Cells wereharvested at various times post-infection and the number of infectiousvirus particles was determined on 293 cells by standard plaque assay(Yu, D.-C. et al., 1999, Cancer Res. 59:4200-4203.). Although theinitial rate of increase of CV7137 in cells treated with CV787 anddocetaxel was similar to that of cells treated with CV787 alone, aplateau was reached for CV787 at approximately 72 post-infection and atapproximately 96 hours post-infection for CV787 and docetaxel. Cellstreated with CV787 and docetaxel produced 30,000 PFU per cell, while thecells infected with CV787 alone generated about 15,000 PFU per cell.Thus, docetaxel does not inhibit CV787 replication, but actuallyincreases virus replication efficiency. A similar results was obtainedin a parallel study with paclitaxel.

Combination of Taxane and CV787 Increases the p53 Expression

To address the synergistic mechanism behind combination treatment, LNCaPis cells were treated with various agents and the expression ofapoptotic related protein markers were compared by Western blot. Thetreatments for LNCaP cells were grouped as (1) docetaxel alone at 6.0nM, (2) CV787 alone at, MOI 0.5, and (3) CV787 (MOI=0.5) and docetaxel(6.0 nM) together. For each treatment group, cells were collected atdifferent time points and subjected to various antibodies by Westernblot. Under these experimental conditions, in the first 48 hours aftertreatment, the combination of CV787 and taxane increased p53 expressionup to 2 to 8-fold compared to virus alone or drug alone at 24 or 48hours.

In contrast, the apoptotic indicators caspase-7 andpoly-ADP-ribose-polymerase did not show a significant change. Inaddition, the combination of CV787 and taxane did not change Fas/Fas-Lor Bcl-2, Bcl-XL, and Bax expression compared to the single agent group.Previously, it was suggested that paclitaxel-induced apoptosis was notmediated by Bcl-2 family change. In the current study, we did notobserve a significant change of Bcl-2 expression in the cells treatedwith docetaxel alone, CV787 alone, or docetaxel and CV787. Liu and Steinhas reported that paclitaxel treated LNCaP cells experienced alterationIn bcl-X_(L) and Bak expression. However, under our condition of lowconcentration of docetaxel, there was no dramatic change detected. Fromthe increased p53 expression, p53-dependent apoptosis may play a majorrole in the synergy of CV787 and taxane.

Synergistic Efficacy of Taxane with CV787 In Vivo

The in vivo antitumor efficacy of CV767 in combination with taxane wasassessed in the LNCaP mouse xenograft model. We have shown previouslythat a single intravenous administration of CV787 at 1×10¹¹ particlesper animal can eliminate subcutaneous xenograft tumors in 6 weeks (Yu,D.-C. et al. (1999) supra. This data was extended using studies upthrough 10 weeks. Established human prostate tumors (LNCaP cells) weretreated with either vehicle, CV787 (1×10¹⁰ particles per animal),paclitaxel (20 mg/kg), or both CV787 and paclitaxel. For the combinationtreatment, animals were intravenously injected with either CV787 orvehicle, and twenty-four hours later, paclitaxel was administeredintraperitoneally (i.p.) daily for four days. The tumor volume datashows that there was a significant decrease in tumor volume betweencontrol and all treatment groups. In this study, single doses of CV787or 4 doses of paclitaxel over four days were effective in producingpartial tumor regression 7 weeks or 2 weeks after treatment, whereas thecombination produced a near complete regression within 2 weeks. Fourweeks after treatment, relative tumor volume decreased to 3% of baseline(from 418 mm³ to 14 mm³) for the combination treatment group and 31% ofbaseline for the paclitaxel group, but increased to 216% of baseline forthe vehicle-treated group and 162% of baseline for the CV787 group.These changes were statistically significant by Students t-test (p<0.05)for the comparison of the combination treatment of CV787 with paclitaxelto any of the vehicles, CV787 or paclitaxel, alone. Additionally, serumPSA levels in mice injected with vehicle increased, whereas the levelsin mice injected with CV787 and paclitaxel decreased to ˜2% of theirstaffing values within 4 weeks.

Combination therapy showed more than additive effect (e.g. synergy) ontumor growth inhibition. On day 21, there was 4.4-fold improvement inanti-tumor activity in the combination group when compared with theexpected additive effect. At this time point, CV787 alone or paclitaxelalone inhibited tumor growth by 20% or 70%, respectively (fractionaltumor volume, 0.815 mm³ and 0.287 mm³ respectively) when compared withthe control group. With time, there was a progressive improvement inanti-tumor activity. On day 42, CV787 and paclitaxel combination groupshowed a 9.2-fold higher inhibition of tumor growth over additive effect(expected fractional tumor volume). These data demonstrated asynergistic efficacy between CV787 and paclitaxel in LNCaP xenografts.

A synergistic effect was also observed in the combination treatment ofxenograft tumors with CV787 and docetaxel. Results from LNCaP prostatetumor xenografts treated with CV787 and docetaxel, both administeredintravenously: in the combination treatment group, animals wereintravenously injected with docetaxel (5.0 mg/kg) on day 2, day 5 andday 8, following a single intravenous injection of CV787 (1×10¹⁰particles per animal) on day 1. Both CV787 and docetaxel appear to beeffective in producing stabilization of tumor growth in the LNCaP mousemodel, whereas a combination of the two produce a complete regressionwithin 5 weeks (FIG. 613). Analysis on fractional tumor volume,indicated a synergistic effect between CV787 and docetaxel in LNCaPxenografts. For example, on day 42, CV787 and docetaxel combinationgroup showed a 6.4-fold higher inhibition of tumor growth over anadditive effect.

To further investigate the dose range for CV787 treatment in combinationwith docetaxel, we fixed the dose of docetaxel at 12.5 mg/kg and variedthe dose of CV787 from 1×10⁸, 1×10⁹, to 1×10¹⁰ particles per animal.Treatment with CV787 alone or docetaxel alone resulted in tumor growthinhibition. However, the combination of CV787 and docetaxel had thegreatest effect of the treatments tested. Complete regression wasachieved in the animals treated with docetaxel and CV787 at a dose ofeither 1×10¹⁰, 1×10⁹, or 1×10⁸ particles. Synergy of anti-tumor activitywas also evident using 1×10⁷ particles per animal but completeregression was not observed. These changes were statisticallysignificant by the Student's t-test for the comparison of combinationtreatment of CV787 and docetaxel to any of the vehicle, CV787 alone, ordocetaxel alone treatments, with no statistical difference between thethree combination treatment groups. Recall the complete response dose ofCV787 alone is 1×10¹¹ particles per animal (Yu, D.-C. et al., 1999,Supra. Thus, the combination of CV787 and docetaxel produces a completeresponse with 1000-fold less virus, compared to the use of CV787 alone.

Virus replication within LNCaP tumors was documented byimmunohistochemical staining of tumor sections using polyclonalantibodies to Adenovirus type 5 (Chen, Y. et al., 2000, Hum. Gone Ther.11:1153-1567.) No evidence of virus replication was found in the tumorstreated with either vehicle or paclitaxel, whereas evidence of necrosisand multifocal inflammation was observed in a small portion of tumorstreated with paclitaxel. In the CV787-treated tumors, while positivelystained cells were visible throughout the tumors, infected cells werepredominantly located near the tumor vasculature. The most intriguingphenomena were in the samples treated with both the virus andpaclitaxel. While few virus-infected cells were detected, most of thecells in the sections were empty and virtually devoid of cellularcontent. The remaining cells were much smaller and appeared to haveundergone a morphological change.

Tumor cells were also tested for apoptosis using the M30 CytoDEATH™detection kit, which recognizes a specific caspase cleavage site withincytokeratin 18 in early events of apoptosis. Three tumors from eachgroup, CV787 alone, paclitaxel, or both CV787 and paclitaxel, wereanalyzed 9 days after the start of dosing. Few apoptotic cells weredetected in the paclitaxel-treated tumor, while a significant amount ofapoptotic cells along the blood vessel were present in theCV787-infected tumors. However, combination treatment produced moreapoptotic cells than in the any of the other samples. In conclusion, theimmunohistochemical analysis of CV787 treated tumors suggests that bothvirus replication-dependent cytolysis and apoptosis contribute to theantitumor effect of CV787 and taxane.

Finally, and of clinical significance are two other results. First,healthier animals, characterized by body weight, were observed in thecombination treatment group as compared to groups treated with eitheragent alone. Of particular interest is the transient weight loss usingdocetaxel alone, from which animals are protected from by the use ofCV787 in combination with docetaxel. Indeed, animals treated with bothCV787 and taxotere gain 24% more weight than untreated control animals(Table 2). Second, formal toxicology studies in Balb/C mice failed toshow synergistic toxicity from the combined use of docetaxel and CV787.

Example 2 In Vitro Treatment of HepG2 and Hep3B Tumor Cells withReplication Competent Target Cell-Specific Adenoviral Vector CV790 andChemotherapy

Regimen for In Vitro Study of Adenoviral Vector and ChemotherapeuticAgent

A preliminary experiment was performed to compare three differentprotocols: Adding virus first, drug first or virus and drug together(FIGS. 15-17). HepG2 and Hep3B cells were treated with 10 ng/mldoxorubicin and 0.01 MOI of CV790. FIG. 15 shows a synergistic effect inthe panel of virus infection first. Virus first indicates administrationof the virus about 10-14 hrs before drug application. Drug firstindicates administration of the chemotherapeutic agent 10-14 hrs beforevirus infection FIG. 16. The results of administration of adenovirusvector and drug together are shown in FIG. 17. For the combination ofCV790 and doxorubicin, virus first administration resulted in thegreatest killing of liver cancer cells. This order of administration wasnot the most effective for CV787 combined with paclitaxel (TAXOL™) ordocetaxel (TAXOTERE™).

In order to study the killing effect of virus and drug on liver cancercells, an in vitro cell viability study (MTT assay) was carried outusing chemotherapeutic agents and the CV790 adenovirus on HepG2 andHep3B hepatoma cells. Protocols for cell growth and MTT assay were asdescribed as in Example 1. CV790 was constructed according to methodsknown in the art with the E1A and E1B genes under the control of theα-fetoprotein promoter (approximately 0.8 kb), with an intact E3 region.The structure of CV790 can be summarized as AFP/E1A, AFP/E1B, E2, E3,E4. The hepatoma cells were grown in well plates, then treated withCV790 and various chemotherapeutic agents, as shown in FIGS. 15-22.After treatment, cells were incubated with MTT and cell viability atdifferent time points from days 2-10 were compared. The MTT assaydetermines the number of cancerous cells still viable after treatmentwith the CV790/chemotherapy combination. Dead cells are equal to 1−thepercentage of viable cells.

The following is the list of chemotherapeutic agents (drugs) and thesources for the drugs used in this study.

-   1. 5-Fluorouracil, (Sigma, St. Louis, Mo.) catalog number F-6627-   2. Doxorubicin hydrochloride, (Sigma, St. Louis, Mo.) catalog number    D-1 515-   3. Cis-platinum (if)-diammine dichloride (cisplatin), (Sigma, St.    Louis, Mo.) catalog number P-4394-   4. 5-azacytidine, (Sigma, St. Louis, Mo.) catalog number A-2385-   5. Mitomycin C, (Sigma, St. Louis, Mo.) catalog number M-0505-   6. TAXOL™, (Mead Johnson oncology products, New Jersey) catalog    number C 0015-3475-30-   7. Gemcitabine, (Lilly, Ind.) catalog number nC 0002-7501-01-   8. Etoposide, (Bristol Laboratories, New Jersey) catalog number nC    0015-3095-20-   9. Mitoxantrone, (Immunex Corp., Seattle, Wash.), catalog number NDC    58406-640-03    Screening of Chemotherapeutic Agents for Synergistic Effects with    CV790

The cytotoxicity of different chemotherapeutic agents combined withCV790 in Hep3B and HepG2 hepatoma cells were tested using themethodology described in Example 1 and above, with virus added beforetreatment with chemotherapeutic agent. The results are shown in FIGS.18-22 and summarized in Table 6, below. These results correspond to thevirus first regimen described above. Doxorubicin, mitomycin C,mitoxantrone, cisplatin, gemcitabine, 5-azacytidine, etoposide andTAXOL™ displayed synergistic effects of cytotoxicity when combined withCV790 compared to the cytotoxicity of the drug or virus alone.5-Fluorouracil did not show synergistic effects with respect to virusand chemotherapy alone. For the experiments summarized in Table 6 theadministered dose of CV790 was either MOI 0.1 or 0.01, as shown in theFigures. The chemotherapeutic agents were administered in the followingamounts: doxorubicin (50 ng/ml); cisplatin (10 μg/ml); taxol (6.5ng/ml); 5-fluorouracil (100 μg/ml); mitoxantrone (100 nM); mitomycin C(10 μg/ml); 5-azacytidine (10 μg/ml); etoposide (1 μg/ml); andgemcitabine (50 ng/ml). TABLE 6 Synergistic Effects ofCV790/Chemotherapeutic Combinations Chemothera- peutic SYN- Virus Cellline agent Class of agent ERGY CV790 HepG2, Hep3B 5-FluorouracilAntimetabolites No CV790 HepG2, Hep3B 5-Azacytidine Antimetabolite Yes(DNA damaging agent) CV790 HepG2, Hep3B Cisplatin Alkylating agent Yes(Plantinum- containing agents) CV790 HepG2, Hep3B DoxorubicinAntibiotics Yes (anticycline) CV790 HepG2, Hep3B TAXOL ™ Plant alkaloidsYes (paclitaxel) CV790 HepG2, Hep3B Etoposide Plant alkaloids Yes CV790HepG2, Hep3B Gemcitabine Antimetabolite Yes (DNA damaging agent) CV790HepG2, Hep3B Mitomycin C Antibiotics Yes CV790 HepG2, Hep3B MitoxantroneAntibiotics Yes (anticycline)

Example 3 In Vitro Treatment of HepG2 and Hep3B Tumor Cells withReplication-Competent AFP-Producing Cell-Specific Adenoviral VectorCV790 and Combination Chemotherapy

In addition to screening single chemotherapeutic agents co-administeredwith replication-competent target cell-specific adenoviral vectors, ascreen was completed of a number of combination chemotherapy regimenswhich were co-administered with CV790, a hepatoma specific adenoviralvector. Examples of such combination or multiple drug chemotherapyregimens can be found in Table 2. The protocols for the administrationof the drugs and virus were as described in Examples 1 and 2, as was themonitoring of cell viability by MTT assay. The regimen followed was thevirus first regimen. A range of drug concentrations were tested.

Treatment of hepatoma cells (Hep3B and HepG2) with a combination ofmultiple chemotherapy drugs plus CV790 showed a synergistic enhancementof cytotoxicity toward the hepatoma cells compared to the treatment ofthe hepatoma cells with either the virus alone or the multiple drugcombination alone. Results are summarized in Table 7 below. TABLE 7Synergistic effects of CV790/Combination Chemotherapeutics CHEMOTHERA-PEUTIC CLASS OF SYN- VIRUS CELL LINE AGENT AGENT ERGY CV790 HepG2, Hep3BDoxorubicin & Anticycline Yes Cisplatin antibiotics & Plantinum-containing agents CV790 HepG2, Hep3B Doxorubicin & Anticycline YesMitomycin C antibiotics CV790 HepG2, Hep3B Doxorubicin & Anticycline YesMitoxantrone antibiotics CV790 HepG2, Hep3B Doxorubicin & AnticyclineYes TAXOL ™ antibiotics & Plant alkaloids

Example 4 Treatment of Prostate Tumor Xenografts with CV787 andChemotherapeutic Agents

After a synergistic effect was observed in vitro for the suppression oftumor cell growth with combinations of CV787 and a number ofchemotherapeutic agents, a subset of these agents were examined forevidence of synergistic results in suppressing tumor growth in vivo. Invivo studies indicated that the combination of CV787 with paclitaxel ordocetaxel could eliminate tumors within 2-4 weeks with ten-fold lessvirus (1×10⁷ particles per mm³ for intratumoral administration, 1×10¹⁰particle per animal for intravenous administration) compared to apreviously effective dose for virus alone. Yu et al. (1999) Cancer Res.59:4200. Below are described detailed examples for CV787 and paclitaxeland CV787 and docetaxel.

Six to eight week old athymic Balb/c nu/nu mice were obtained fromSimonson Laboratories (Gilroy, Calif.) and acclimatized to laboratoryconditions one week prior to tumor implantation. Xenografts wereestablished by injecting 1×10⁶ LNCaP cells subcutaneously near the smallof the back suspended in 100 μl of RPMI 1640 and 100 μl of maltrigel(Collaborative Biochemical Products). When tumors reached between 400mm³ and 600 mm³, mice were randomized in groups of four each to receiveeither 1×10¹⁰ particles of CV787 at day 1 via the tail vein orpaclitaxel, 20 mg/kg intraperitoneally (i.p.) daily for 4 days startingat day 2, versus controls treated with normal saline 0.1 ml i.v. at day1 and then i.p. for 4 days. In addition, another group of mice receivedthe combination of CV787 and paclitaxel at the same doses and scheduleas above. CV787 was diluted in lyophilized buffer and injected into tailvein in a volume of 0.1 ml using a 28-gauge needle. The dose and routeof administration of paclitaxel were selected according to studies innude mice by Riondel et al., (1986) Cancer Chemother Pharmacol. 17:137.These authors conducted acute toxicity studies of paclitaxel in nudemice and selected the unit dose of 12.5 mg/kg daily, being 1/20th of theLD50 dose (lethal dose for 50% of animals). Tumors were measured weeklyin two dimensions by external caliper and volume was estimated by theformula [length (mm)×width (mm)²]/2. Animals were humanely killed whentheir tumor burden became excessive. Serum was harvested weekly byretro-orbital bleed. The difference in mean tumor volume betweentreatment groups was compared for statistical significance using theunpaired, two-tailed, t-test. Blood samples were collected at varioustime points for determining prostate-specific antigen (PSA). The levelof PSA is directly related to tumor size, and tumor regression isaccompanied by a fall in PSA levels.

Anti-Tumor Efficacy of the Combined Therapy of IntratumorallyAdministered CV787 with Paclitaxel

The in vivo antitumor efficacy of intratumorally administered CV787 andthe interaction of CV787 in the combination with paclitaxel was assessedin the LNCaP mouse xenograft model as described above. The followingtreatments were administered (n=6 per treatment group):

Vehicle (negative control).

CV787 (active control) at a dose of 1×10⁷ particles per mm³ of tumor, atday 1.

Paclitaxel at a dose of 15 mg/kg of animal weight, starting at day 2,daily for four days.

CV787 (1×10⁷ particles per mm) and paclitaxel (15 mg/kg), scheduled asabove.

All treatment groups received identical injections of the active agentor vehicle control into both the tumor and peritoneum. Tumor volume wasmeasured just before the injection of test articles and once a week for6 weeks thereafter.

The following changes in average tumor volumes were measured 6 weeksafter treatment. Average tumor volume increased in vehicle-treatedanimals from 425 mm³ to 983 mm³ (231% of baseline) 6 weeks aftertreatment and in the paclitaxel group from 405 mm³ to 630 mm³ (166% ofbaseline) FIG. 23. Tumor volumes in the CV787 1×10⁷ particles/mm³ groupdropped from 419 to 379 mm³ (90% of baseline) whereas the average tumorvolume in the combination treatment group of CV787 with paclitaxeldecreased from 413 mm³ to 45 mm³ (11% of baseline) within six weeksafter treatment. These changes were statistically significant byStudent's t-test for the comparison of combination treatment of CV787with paclitaxel to any of the vehicles, CV787 alone or paclitaxel alonetreatment. It is suggested that the combination of CV787 with paclitaxelproduces a synergistically enhanced anti-tumor efficacy, more effectivethan virus treatment alone or paclitaxel treatment alone.

Anti-Tumor Efficacy of the Combined Therapy of IntravenouslyAdministered CV787 with Paclitaxel

In vivo studies of intravenously administered CV787 in conjunction withpaclitaxel or docetaxel were performed in the same mouse xenograft modelas used for the intratumoral injection study. All test articles wereadministered via tail vein except that paclitaxel was injectedintraperitoneally into animals.

The efficacy of intravenously administered CV787 and paclitaxel wasassessed as described above. The following treatments were administeredin this study:

Vehicle (negative control).

CV787 (active control) at a dose of 1×10¹⁰ particles per animal at day1.

Paclitaxel at a dose of 20 mg/kg of animal weight, starting at day 2,daily for 4 days.

CV787 (1×10 μl particles/animal) and paclitaxel (20 mg/kg), scheduled asabove.

Tumor volumes were measured just before the injection of test articlesand once a week for 10 weeks thereafter.

In this study, single doses of CV787 and paclitaxel both appeared to beeffective in producing tumor regression in the LNCaP mouse model,whereas the combination produced a complete regression in 4 weeks FIG.25. Four weeks after treatment, relative tumor volume decreased to 3% ofbaseline (from 418 mm³ to 14 mm³) for the combination treatment groupand 216% of baseline for the vehicle-treated group, 31% of baseline forthe paclitaxel group and 162% of baseline for the CV787 group. Ten weeksafter treatment, 100% of the animals in the combination therapy groupwere tumor free, and animals were followed for 90 days without tumorregrowth. Relative tumor volume in the CV787-treated group decreased to28% of baseline, while the tumors in the paclitaxel-treated groupprogressively grew back to 149% of baseline. This result indicated thatpaclitaxel alone could not cure cancers in this xenograft model. CV787appeared to be highly effective and virus alone took a relatively longperiod of time to cure cancer at this dose level. However, thecombination of CV787 and paclitaxel effectively eliminated tumors within4 weeks after administration. In summary, the combination of paclitaxelwith intravenously administered CV787 was far more effective thanchemotherapy or virus treatment alone.

FIG. 24 depicts the change in tumor growth upon varying does ofpaclitaxel (TAXOL™) and CV787 combined with paclitaxel (TAXOL™).paclitaxel (TAXOL™) at 10 mg/kg has approximately the same efficacy overa 5 week period as does paclitaxel (TAXOL™) at 2 mg/kg when combinedwith CV787 (1×10¹⁰ particles). Each of these treatments merely arreststumor growth while not actually causing and regression of the tumor. Adose of 2 mg/kg of paclitaxel (TAXOL™) alone, however, leads toprogressive enlargement of the tumor. A combination of paclitaxel(TAXOL™) at 10 mg/kg combined with a 1×10¹⁰ particle dose of CV787,however, leads to complete suppression of tumor growth by the third weekof the in vivo trial.

Anti-Tumor Efficacy of the Combined Therapy of IntravenouslyAdministered CV787 and Docetaxel

The efficacy of intravenously administered CV787 and docetaxel was alsoassessed as described above. All test articles were administered intoanimals via tail vein. The following treatments were administered inthis study:

Vehicle (negative control).

CV787 (active control) at a dose of 1×10¹⁰ particles per animal at day1.

Docetaxel at a dose of 10 mg/kg of animal weight, starting at day 2,daily for 4 days.

CV787 (1×10¹⁰ particles/animal) and paclitaxel (10 mg/kg), scheduled asabove.

Tumor volumes were measured just before the injection of test articlesand once a week for 6 weeks thereafter.

In this study, single dose of CV787 and docetaxel both appeared to beeffective in producing tumor regression in the LNCaP mouse model,whereas the combination produced a complete regression within 4 weeks.Four weeks after treatment, relative tumor volume decreased to 2% ofbaseline for the combination treatment group and 226% of baseline forthe vehicle-treated group, 49% of baseline for the docetaxel group and132% of baseline for the CV787 group. These changes were statisticallysignificant by the Student's t-test for the comparison of combinationtreatment of CV787 and docetaxel to any of the vehicle, CV787 alone ordocetaxel alone treatment. It is suggested that the combination of CV787and docetaxel produces an enhanced anti-tumor efficacy, much better thanvirus alone or docetaxel alone.

An alternate presentation of these data are found in FIG. 28 in whichthe data are reported as tumor volumes.

Following the successful treatment of the LNCaP xenografts with theabove-described method, the dosage of docetaxel was decreased by 50% to5 mg/kg and the CV787 dose was maintained at 1×10¹⁰ particles. As shownin FIG. 29 significant regression of the tumor is observed for theCV787/docetaxel combination therapy by week 3. At week 4 the tumorvolume is less than the tumor volume which remains steady for theremainder of the experiment. The minimum tumor volume for docetaxelalone, approximately 50% of the original tumor volume, is reached byweek 1, however, in the following weeks tumor growth resumes and by week6 has reached the starting tumor volume. Treatment with a tenfold higherdose of CV787 (1×10¹¹ particles) is significantly more effective thanthe lower dose of CV787 (1×10¹⁰ particles) or docetaxel alone, but isslower to regress tumor growth and even at week 6 does not equal thereduction in tumor volume as the combination of CV787 (1×10¹⁰ particles)and docetaxel (5 mg/kg).

In summary, in vivo studies showed that direct intratumoral orintravenous injection of CV787 in conjunction with paclitaxel ordocetaxel has an enhanced anti-tumor efficacy, resulting in asignificantly lower tumor burden observed in the combination treatment.The virus dose in the combination treatment was 10-fold lower than ourpreviously effective dose for virus treatment alone, 1×10¹¹ particlesand a hundred percent of treated animals had complete tumor regressionwithin 4 weeks in the intravenous administration regimen. These dataprovide supportive evidence for the potential development of acombination clinical regimen of CV787 with paclitaxel or docetaxel forclinical treatment of prostate cancer.

A number of other chemotherapeutic agents were screened for synergisticeffect when combined with CV787 for the in vivo treatment of cancer.Results are summarized in Table 8, below, and representative data shownin FIGS. 23, 25-27. Table 8 also includes data for the CN706 adenoviralvector, a replication-competent prostate cell-specific adenoviralconstruct (see Table 4). CV787 was administered in amounts rangingbetween 1×10⁷ particles/mm³, and 1×10¹¹ particles, as indicated in theFigures. Chemotherapeutic agents were administered in the followingamounts: paclitaxel (TAXOL™; 2 mg/kg to 20 mg/kg as shown); docetaxel(TAXOTERE™; 5-10 mg/kg, as shown); mitoxantrone (3 mg/kg); estramustine(14 mg/kg daily at days 2-5, 7-11, 13-17, and 20-24); cisplatin (4mg/kg); and 5-fluorouracil (30 mg/kg). TABLE 8 Synergistic effects ofAdenovirus/Chemotherapeutic Combinations in vivo Chemothera- peutic Syn-Virus Cell line agent Class of agent ergy CV706 Prostate cancer5-Fluorouracil Antimetabolites Yes xenografts CV787 Prostate cancerCisplatin Alkylating Agent Yes xenografts (Plantinum- containing agents)CV787 Prostate cancer Estramustine Alkylating agent Yes xenografts CV787Prostate cancer Mitoxantrone Antibiotics No xenografts (anticycline)CV787 Prostate cancer TAXOTERE ™ Plant Alkaloids Yes xenografts(docetaxel) CV787 Prostate cancer TAXOL ™ Plant alkaloids Yes xenografts(paclitaxel)

Example 5 Treatment of Hep3B Tumor Xenografts with Replication-CompetentHepatoma Specific CV790 and Doxorubicin and Hepatoma Specific CV890 andDoxorubicin

CV790 is an AFP producing hepatocellular carcinoma specific adenovirus,with E1A and E1B under the control of an identical AFP promoter (827 bp)and enhancer with an E3 region. The CV890 adenovirus construct is also ahepatoma or liver-specific adenoviral mutant with the E1A and E1B genesunder transcriptional control of 827 bp AFP promoter, wherein E1B isunder translational control of EMCV IRES and having an intact E3 region.The structure of CV890 therefore reads as AFP/E1A, IRES/E1B, E2, E3, E4.In vivo studies of the efficacy of combinations of CV790 and doxorubicinand CV890 and doxorubicin were performed according to the protocolsdescribed in detail in Example 4, with minor alterations which aredescribed below.

Xenografts in the study of CV790 and CV890 combined withchemotherapeutic agents utilized liver carcinoma Hep3B cells, instead ofLNCaP prostate carcinoma. Virus, CV790 or CV890, was administered by asingle intravenous injection of 1×10¹¹ particles through the tail veinsof the nude mice. One day after virus delivery, a single dose ofdoxorubicin was given to each animal by i.p. injection. The doxorubicindose was 10 mg/kg for both doxorubicin alone and doxorubicin combinedwith virus treatments. Tumor volume was measured once a week for sixweeks according to the protocol in Example 4.

Both CV790/doxorubicin and CV890/doxorubicin treatment of the hepatomashowed synergistic results. Four weeks after treatment with eitherCV790/doxorubicin or CV890/doxorubicin the relative tumor volume wasless than 10%. Unlike mice treated with either virus alone ordoxorubicin alone, after week 4, the relative tumor volume did notincrease for either the either CV790/doxorubicin or CV890/doxorubicintreated mice. At week 6 in the control mice, the relative tumor volumewas approximately 1000% in the CV790 study and approximately 600% in theCV890 study 4 weeks after treatment. The relative tumor volumes of micetreated with virus alone were 250% (CV790) and 520% (CV890) while therelative tumor volumes for mice treated with doxorubicin alone were 450%with 280% in the CV790 study and 500% in the CV890 study. These resultsare shown in FIG. 30 (CV790/doxorubicin) and FIG. 31(CV890/doxorubicin).

Example 6 In Vitro Treatment of Tumor Cells with Combined TargetCell-Specific Adenoviral Vector CV787 and Radiation Therapy

LNCaP prostate carcinoma cells were pre-seeded in 96 well plates in theRPMI medium at 10,000 cells per well. After infection with CV787 (0.01MOI) according to above described protocols (Example 1), the cells wereincubated at 37° C. with 5% CO₂ for 24 hours, and then irradiated asmonolayers using Cesium 137 gamma rays (used for all radiation studies)at a dose of 2 Gy. An MTT assay as described in Example 1 was performedto determine cell viability (1−% of viable cells=dead cells). Theresults are shown in FIGS. 10; 32; 33; 34; 35; and 36. The results showthat the combined adenoviral/radiation treatment is synergisticallyenhanced over treatment with either virus or radiation alone.

FIG. 33 summarizes the results of a comparison of the treatment of LNCaPprostate carcinoma cells with 6 Gy radiation combined with CV787 (MOI0.1), 6 Gy radiation alone, CV787 treatment alone, or no treatment.Protocols for the treatment are as described above for CV787 and 2 Gyradiation. Synergistic results were observed for the combined treatmentof adenovirus and radiation compared to either treatment alone.

In FIG. 32 the same procedure was followed as those described above withthe treatment consisting of CV787 (MOI 0.1) and 6 Gy radiation, exceptthat the virus was added 24 hours after LNCaP cells were irradiated. Theresults indicate that virus treatment either before or after irradiationleads to synergy in terms of cell killing.

To establish a dose response curve, LNCaP cells were prepared andtreated as described above, with CV787 (MOI 0.01) administered first,followed after 24 hours with varying doses of radiation. Separate cellcultures were irradiated with an increasing dose of radiation startingat 0 Gy, up to 8 Gy (CV787 was kept at the same level of multiplicity ofinfection of 0.01), then 6 days after irradiation, the cells weresubjected to a MTT assay as described above in Example 1. FIG. 36 showsthe resulting dose response curve, with nearly 100% cell death at day 6for an 8 Gy dose of radiation.

To determine the effect of radiation on the viability of thereplication-competent target cell-specific adenoviral vectors, virusyield was measured according to the protocol described in Example 8.LNCaP prostate carcinoma cells were seeded in well plates as describedin Example 1 and above, then treated with either radiation (6 Gy)followed by administration of CV787 (MOI 0.1) 24 hours later or treatedwith CV787 (MOI 0.1) followed 24 hours later by irradiation (6 Gy), asdescribed above in this example. These results were compared to thevirus yield determined in LNCaP cells treated with CV787 (MOI 0.1)alone. In both cases, with either radiation administered first FIG. 34or virus administered first FIG. 35, the virus yield over time iscomparable to the virus yield in LNCap cells which are not treated withradiation. These results indicate that the combination treatmentproduces more virus than virus alone.

Example 7 Construction of a Replication-Competent Adenovirus VectorComprising an AFP-TRE and an EMCV IRES

The encephalomyocarditis virus (ECMV) IRES as depicted in Table 12 wasintroduced between the E1A and E1B regions of a replication-competentadenovirus vector specific for cells expressing AFP as follows. Table 12shows the 519 base pair IRES segment which was PCR amplified fromNovagen's pCITE vector by primers A/B as listed in Table 9. A 98 basepair deletion in the E1A promoter region was created in PXC.1, a plasmidwhich contains the left-most 16 mu of Ad5. Plasmid pXC.1 (McKinnon(1982) Gene 19:33-42) contains the wild-type left-hand end of Ad5, fromAdenovirus 5 nt 22 to 5790 including the inverted terminal repeat, thepackaging sequence, and the E1a and E1b genes in vector pBR322. pBHG10(Bett. et al. (1994) Proc. Natl. Acad. Sci. USA 91:8802-8806; MicrobixBiosystems Inc., Toronto) provides the right-hand end of Ad5, with adeletion in E3. The resultant plasmid, CP306 (PCT/US98/16312), was usedas the backbone in overlap PCR to generate CP624. To place a Sal1 sitebetween E1a and E1b, primers C/D, E/F (Table 9) were used to amplifyCP306, plasmid derived from pXC.1 and lacking the E1a promoter. Afterfirst round PCR using CP306 as template and primers C/D, E/F, theresultant two DNA fragments were mixed together for another round ofoverlapping PCR with primers C/F. The overlap PCR product was cloned byblunt end ligation to vector. The resultant plasmid, CP624 (Table 10),contains 100 bp deletion in E1a/E1b intergenic region and introducesSal1 site into the junction. On this plasmid, the endogenous E1apromoter is deleted, and the E1a polyadenylation signal and the E1 bpromoter are replaced by the Sal1 site. Next, the Sal1 fragment of CP625was cloned into the Sal1 site in CP624 to generate CP627 (Table 10).CP627 has an EMCV IRES connecting adenovirus essential genes E1a andE1b. In CP627, a series of different tumor-specific promoters can beplaced at the PinA1 site in front of E1a to achieve transcriptionalcontrol on E1 expression. TABLE 9 Primer Sequence Note A.5′-GACGTCGACTAATTCCGGTTATTTTCCA For PCR EMCV IRES, GTCGAC is a Sa1Isite. B. 5′-GACGTCGACATCGTGTTTTTCAAAGGAA For PCR EMGV IRES, GTCGAC is aSa1I site. C. 5′-CCTGAGACGCCCGACATCACCTGTG Ad5 sequence to 1314 to 1338.D. 5′-GTCGACCATTCAGCAAACAAAGGCGTTAAC Antisense of Ad5 sequence 1572 to1586. GTCGAC is a Sa1I site. Underline region overlaps with E. E.5′-TGCTGAATGGTCGACATGGAGGGTTGGGAG Ad5 sequence 1714 to 1728. GTCGAC is aSa1I site. Underline region overlaps with D. F.5′-CACAAACCGCTCTCCACAGATGCATG Antisense of Ad5 sequence 2070 to 2094.

For generating a liver cancer-specific virus, an about 0.8 kb AFPpromoter fragment as shown in Table 14 was placed into the PinA1 site ofCP627 thereby yielding plasmid CP686. Full-length viral genomes wereobtained by recombination between CP686 and a plasmid containing a rightarm of an adenovirus genome. The right arms used in virus recombinationwere pBHGE3 (Microbix Biosystems Inc.), containing an intact E3 region,and pBHG11 or pBHG10 (Bett et al. (1994) containing a deletion in the E3region.

The virus obtained by recombination of CP686 with a right arm containingan intact E3 region was named CV890. The virus obtained by recombinationof CP686 with a right arm containing a deleted E3 region (pBHG10) wasnamed CV840. The structure of all viral genomes was confirmed byconducting PCR amplifications that were diagnostic for the correspondingspecific regions.

Therefore, adenovirus vector designated CV890 comprises 0.8 kb AFPpromoter, E1A, a deletion of the E1A promoter, EMCV IRES, E1B a deletionof the E1B promoter and an intact E3 region. Adenovirus vector CV840comprises AFP promoter, E1A, a deletion of the E1A promoter, EMCV IRES,E1B, a deletion of the E1B promoter and a deleted E3 region. TABLE 10Plasmid designation Brief description CP306 An E1A promoter deletedplasmid derived from pXC.1 CP624 Overlap PCR product from CP306 togenerate 100 bp deletion and introduce a Sal1 site at E1A and E1Bjunction; E1A and E1B promoter deleted in E1A/E1B intergenic region.CP625 EMCV IRES element ligated to PCR-blunt vector (Invitrogen pCR ®blunt vector). CP627 IRES element derived from CP625 by Sal1 digestionand ligated to CP624 Sal1 site placing IRES upstream from E1B. CP628Probasin promoter derived from CP251 by pinAl digestion and cloned intopinAl site on CP627. CP629 HCMV IE promoter amplified from pCMV beta(Clontech) with pinAl at 5′ and 3′ ends ligated into CP627 pinAl site.CP630 A 163 bp long VEGF IRES fragment (Table 1) cloned into the Sal1site on CP628. CP686 AFP promoter from CP219 digested with pinAl andcloned into pinAl site on CP627.

Example 8 Construction of a Replication-Competent Adenovirus Vector witha Probasin TRE and an EMCV IRES

The probasin promoter as shown in Table 14 was inserted at the PinAIsite of plasmid CP627 (see Example 8) to generate CP628, which containsa probasin promoter upstream of E1A and an EMCV IRES between E1A andE1B. Full-length viral genomes were obtained by recombination betweenCP628 and a plasmid containing a right arm of an adenovirus genome. Theright arms used in virus recombination were pBHGE3, containing an intactE3 region, and pBHG11 or pBHG10 containing a deletion in the E3 region.The structure of all viral genomes was confirmed by conducting PCRamplifications that were diagnostic for the corresponding specificregions.

Therefore, adenovirus designated CV 834 comprises probasin promoter,E1A, a deletion of the E1A promoter, EMCV IRES, E1B, a deletion of theE1B endogenous promoter and a deleted E3 region.

Example 9 Construction of a Replication-Competent Adenovirus Vector witha hCMV-TRE and an EMCV IRES

The hCMV immediate early gene (IE) promoter from plasmid CP629,originally derived from pCMVBeta (Clonetech, Palo Alto) was inserted atthe PinAI site of plasmid CP627 (see Example 8) to generate CP629,containing a CMV IE promoter upstream of E1A and an IRES between E1A andE1B. Full-length viral genomes were obtained by recombination betweenCP629 and a plasmid containing a right arm of an adenovirus genome. Theright arms used in virus recombination were pBHGE3, containing an intactE3 region, and pBHG11 or pBHG10 containing a deletion in the E3 region.The structure of all viral genomes was confirmed by conducting PCRamplifications that were diagnostic for the corresponding specificregions.

Therefore, adenovirus vector designated CV835 comprises hCMV-IEpromoter, E1A, a deletion of the E1A promoter, EMCV IRES, E1B a deletionin the E1B endogenous promoter and a deleted E3 region. CV835 lacks thehCMV enhancer and is therefore not tissue specific. By adding the hCMVIE enhancer sequence to CV835, the vector is made tissue specific.

Example 10 Comparison of Dual TRE Vectors with SingleTRE/IRES-Containing Vectors

Two liver cancer-specific adenovirus vectors, CV790 and CV733 (alsodesignated CN790 and CN733, respectively), were generated andcharacterized. See PCT/US98/04084. These viruses contain two AFP TREs,one upstream of E1A and one upstream of E1B. They differ in that CV790contains an intact E3 region, while the E3 region is deleted in CV733.Replication of these two viruses was compared with that of the newlygenerated IRES-containing viruses, CV890 and CV840 (see Example 1).

Virus replication was compared, in different cell types, using a virusyield assay as described in Example 4. Cells were infected with eachtype of virus and, 72 hrs after infection, virus yield was determined bya plaque assay. The results indicate that vectors containing an IRESbetween E1A and E1B (CV890 and CV840), in which E1B translation isregulated by the IRES, replicate to similar extents as normal adenovirusand viruses with dual AFP TREs, in AFP-producing cells such as 293 cellsand hepatoma cells. In SK-Hep-1 (liver cells), PA-1 (ovarian carcinoma)and LNCaP cells (prostate cells) the IRES-containing viruses do notreplicate as well as dual TRE or wild-type adenoviruses, indicating thatthe IRES-containing viruses have higher specificity for hepatoma cells.Based on these results, it is concluded that IRES-containing vectorshave unaltered replication levels, but are more stable and have bettertarget cell specificity, compared to dual-TRE vectors.

Example 11 Uroplakin Adenoviral Constructs Containing an EMCV IRES

A number of E3-containing viral constructs were prepared which containeduroplakin II sequences (mouse and/or human) as well as an EMCV internalribosome entry site (IRES). The viral constructs are summarized in Table11. All of these vectors lacked an E1A promoter and retained the E1Aenhancer.

The 519 base pair EMCV IRES segment was PCR amplified from Novagen'spCITE vector by primers A/B: primer A: 5′-GACGTCGACTAATTCCGGTTATTTTCCAprimer B: 5′-GACGTCGACATCGTGTTTTTCAAAGGAA (GTCGAC is a Sa1I site).

The EMCV IRES element was ligated to PCR blunt vector (Invitrogen pCR®blunt vector).

CP1066

The 1.9 kb-(−1885 to +1) fragment of mouse UPII from CP620 was digestedwith AflIII (blunted) and HindIII and inserted into pGL3-Basic fromCP620 which had been digested with XhoI (blunted) and HindIII togenerate CP1066.

CP1086

The 1.9 kb mouse UPII insert was digested with PinAI and ligated withCP269 (CMV driving E1A and IRES driving E1B with the deletions ofE1A/E1B endogenous promoter) which was similarly cut by PinAI.

CP1087

The 1 kb (−1128 to +1) human UPII was digested with PinAI from CP665 andinserted into CP629 which had been cut by PinAI and purified (to eluteCMV).

CP1088

The 2.2 kb (−2225 to +1) human UPII was amplified from CP657 with primer127.2.1 (5′-AGGACCGGTCACTATAGGGCACGCGTGGT-3′) PLUS 127.2.2(5′-AGGACCGGTGGGATGCTGGGCTGGGAGGTGG-3′) and digested with PinAI andligated with CP629 cut with PinAI.

CP627 is an Ad5 plasmid with an internal ribosome entry site (IRES) fromencephelomycarditis virus (EMCV) at the junction of E1A and E1B. First,CP306 (Yu et al., 1999) was amplified with primer pairs 96.74.3/96.74.6and 96.74.4/96.74.5.

The two PCR products were mixed and amplified with primer pairs 96.74.3and 96.74.5. The resultant PCR product contains a 100 bp deletion inE1A-E1B intergenic region and a new Sail site at the junction. EMCV IRESfragment was amplified from pCITE-3a(+) (Novagen) using primers 96.74.1and 96.74.2. The Sail fragment containing IRES was placed into SalI siteto generate CP627 with the bicistronic E1A-IRES-E1B cassette. CP629 is aplasmid with CMV promoter amplified from pCMVbeta (Clontech) with primer99.120.1 and 99.120.2 and cloned into PinAI site of CP627.

CP657 is a plasmid with 2.2 kb 5′ flanking region of human UP II gene inpGL3-Basic (Promega). The 2.2 kb hUPII was amplified by PCR fromGenomeWalker product with primer 100.113.1 and 100.113.2 and TA-clonedinto pGEM-T to generate CP655.

The 2.2 kb insert digested from SacII (blunt-ended) and KpnI was clonedinto pGL3-Basic at HindIII (blunted) and KpnI to create CP657.

CP1089

The 1 kb (−965 to +1) mouse UPII was digested by PinAI from CP263 andinserted into CN422 (PSE driving E1A and GKE driving E1B with thedeletions of E1A/E1B endogenous promoter) cut by PinAI and purified andfurther digested with EagI and ligated with 1 kb (−1128 to +1) humanUPII cut from CP669 with EagI.

CP1129

The 1.8 kb hUPII fragment with PinAI site was amplified from CP657 withprimer 127.50.1 and 127.2.2 and cloned into PinAI site of CP629.

CP1131

CP686 was constructed by replacing the CMV promoter in CP629 with an AFPfragment from CP219. A 1.4 kb DNA fragment was released from CP686 bydigesting it with BssHII, filling with Klenow, then digesting withBglII. This DNA fragment was then cloned into a similarly cut CP686 togenerate CP1199. In CP1199, most of the E1B 19-KDa region was deleted.The 1.8 kb hUPII fragment with PinAI site was amplified from CP657 byPCR with primer 127.50.1 and 127.2.2 and inserted into similarlydigested CP1-199 to create CP1131.

The plasmids above were all co-transfected with pBHGE3 to generate CV874(from CP1086), CV875 (from CP1087), CV876 (from 1088) and CV877 (fromCP1089), CV882 (from CP1129) and CV884 (from CP1131). CP1088, CP1129 andCP1131 were cotransfected with pBHGE3 for construction of CV876, CV892and CV884, respectively by lipofectAMINE (Gibco/BRL) for 11-14 days.pBHGE3 was purchased from Microbix, Inc., and was described previously:The cells were lysed by three freeze-thaw cycles and plaqued on 293cells for a week. The single plaques were picked and amplified byinfection in 293 cells for 3-5 days. The viral DNAs were isolated fromthe lysates and the constructs were confirmed by PCR with primer31.166.1/51.176 for CV876 and primer 127.50.1/51.176 for CV882 and CV884at E1 region and primer 32.32.1/2 for all three viruses at E3 region.TABLE 11 Name Vector Ad 5 Vector E1A TRE E1B TRE E3 CV874 CP1086 pBHGE31.9 kb mUPII IRES intact CV875 CP1087 pBHGE3 1.0 kb hUPII IRES intactCV876 CP1088 pBHGE3 2.2 kb hUPII IRES intact CV877 CP1089 pBHGE3 1.0 kbmUPII 1.0 kb intact hUPII (E1B promoter deleted) CV882 CP1129 pBHGE3 1.8kb hUPII IRES intact CV884 CP1131 pBHGE3 1.8 kb hUPii IRES (E1B intact19-kDa deleted)Viruses are tested and characterized as described above.

Primer sequences: 96.74.1 GACGTCGACATCGTGTTTTTCAAAGGAA 96.74.2GACGTCGACTAATTCCGGTTATTTTCCA 96.74.3 CCTGAGACGCCCGACATCACCTGTG 96.74.4TCTGAATGGTCGACATGGAGGCTTGGGAG 96.74.5 CACAACCGCTCTCCACAGATGCATG 96.74.6GTCGACCATTCAGCAAACAAAGGCGTTAAC 100.113.1 AGGGGTACCCACTATAGGGCACGCGTGGT100.113.2 ACCCAAGCTTGGATGCTGGGCTGGGAGGTGG 127.2.2AGGACCGGTGGGATGCTGGGCTGGGAGGTGG 127.50.1 AGGACCGGTCAGGCTTCACCCCAGACCCAC31.166.1 TGCGCCGGTGTACACAGGAAGTGA 32.32.1 GAGTTTGTGCCATCGGTCTAC 32.32.2AATCAATCCTTAGTCCTCCTG 51.176 GCAGAAAAATCTTCCAAACACTCCC 99.120.1ACGTACACCGGTCGTTACATAACTTAC 99.120.2 CTAGCAACCGGTCGGTTCACTAAACG

Example 12 Construction of a Replication-Competent Adenovirus Vectorwith a Tyrosinase TRE and EMCV IRES

CP621 is a plasmid containing a human tyrosinase enhancer and promoterelements in a PinAI fragment. This fragment is ligated to the PinAI siteon CP627 to generate CP1078. CP1078 is combined with pBHGE3 to generatea new melanoma specific virus, CV859. Table 14 depicts thepolynucleotide sequence of the PinAI fragment which contains atyrosinase promoter and enhancer.

Example 13 Construction of a Replication-Competent Adenovirus Vectorwith a Probasin-TRE and a VEGF IRES

Using a strategy similar to that described in Example 8, the IRESfragment from the mouse vascular endothelial growth factor (VEGF) geneis amplified and cloned into CP628 at the SalI site. Table 12 depictsthe IRES fragment obtainable from vascular endothelial growth factor(VEGF) mRNA. In order to clone this fragment into the E1a/E1b intergenicregion, two pieces of long oligonucleotide are synthesized. The senseoligonucleotide is shown in the table, whereas the second piece is thecorresponding antisense one. After annealing the two together to createa duplex, the duplex is subjected to SalI digestion and the resultingfragment is cloned into the SalI site on CP628. The resulting plasmid,CP630, has a probasin promoter in front of E1a and an VEGF IRES elementin front of E1b. This plasmid is used to construct a prostatecancer-specific virus comprising the VEGF IRES element.

Example 14 Construction of a Replication-Competent Adenovirus Vectorwith an AFP-TRE and a VEGF IRES

Using a strategy similar to Example 8, a PinAI fragment which containsAFP TRE can be obtained. This AFP TRE is cloned into the PinA1 site infront of E1A on CP628 yielding plasmid CP1077. This plasmid has the AFPTRE for E1 transcriptional control and the VEGF IRES element before E1b.CP1077 can be recombined with pBHGE3 to generate a liver-specificadenovirus, designated as CV858.

Example 15 Construction of a Replication-Competent Adenovirus Vectorwith a hKLK2-TRE and a EMCV IRES

Using a strategy similar to Example 1, the TRE fragment from humanglandular kallikrein II as shown in Table 14 was cloned into the PinAIsite in CP627. The resultant plasmid, CP1079, is cotransfected withpBHGE3 to create CV860.

Example 16 Construction of a Replication-Competent Adenovirus Vectorwith a CEA-TRE and a EMCV IRES

Using a strategy similar to Example 1, the TRE fragment fromCarcinembryonic antigen (CEA)(Table 14, SEQ ID NO:______) is used toconstruct virus designated CV873. A PinAI fragment containing theCEA-TRE was cloned into the PinAI site in front of E1A of CP627 for thetranscriptional control. The resultant plasmid CP1080 is used togetherwith pBHGE3 to generate CV873.

Example 17 Adenovirus Vectors with Urothelial Cell-Specific TREs

A number of plasmid constructs were generated as intermediates foradenovirus type 5 (Ad 5) vector constructs. The plasmid constructs werebased on plasmid CP321 (Yu et al., 1999, Cancer Res. 59:4200-4203),which contains a prostate-specific enhancer inserted at a PinAI siteupstream of the E1A gene and at a EagI site upstream of the E1B gene.Constructs were created by inserting various UPII-derived 5′-flankingDNA sequences into the PinAI and EagI sites and removing theprostate-specific enhancer. Characteristics of the plasmid CP669 are E1ATRE 1.0 kb hUPII and E1B TRE 1.0 kb mUPI and lacked the E1A promoter andwhich contained the E1A enhancer. Infectious recombinant adenoviralvectors was produced by co-transfecting 293 cells with the UPII5′-flanking DNA/E1 constructs and an Ad 5 backbone vector (pBHG10 orpBHGE3, Microbix, Inc.) as described in Yu et al. (id.) to produceCV829, which has an intact E3 region.

Example 18 In Vitro Characterization of Melanocyte-SpecificTRE-Containing Adenoviral Constructs

An especially useful objective in the development of melanocytecell-specific adenoviral vectors is to treat patients with melanoma.Methods are described below for measuring the activity of amelanocyte-specific TRE and thus for determining whether a given cellallows a melanocyte-specific TRE to function.

Cells and Culture Methods

Host cells such as, HepG2 (liver); Lovo (colon); LNCaP (prostate); PMEL(melanoma); SKMel (melanoma); G361 (melanoma) and MeWo cells areobtained at passage 9 from the American Type Culture Collection(Rockville, Md.). MeWo cells are maintained in RPMI 1640 medium (RPMI)supplemented with 10% fetal bovine serum (FBS; Intergen Corp.), 100units/mL of penicillin, and 100 units/mL streptomycin. MeWo cells beingassayed for luciferase expression are maintained in 10% strip-serum(charcoal/dextran treated fetal bovine serum to remove T3, T4, andsteroids; Gemini Bioproduct, Inc., Calabasas, Calif.) RPMI.

Transfections of MeWo Cells

For transfections, MeWo cells are plated out at a cell density of 5×10⁵cells per 6-cm culture dish (Falcon, N.J.) in complete RPMI. DNAs areintroduced into MeWo cells after being complexed with a 1:1 molar lipidmixture of N-[1-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium chloride(DOTAP™; Avanti Polar Lipids, AL) and dioleoyl-phosphatidylethanolamine(DOPE™; Avanti Polar Lipids, AL); DNA/lipid complexes are prepared inserum-free RPMI at a 2:1 molar ratio. Typically, 8 μg (24.2 nmole) ofDNA is diluted into 200 μL of incomplete RPMI and added dropwise to 50mmole of transfecting, lipids in 200 μL of RPMI with gentle vortexing toinsure homogenous mixing of components. The DNA/lipid complexes areallowed to anneal at room temperature for 15 minutes prior to theiraddition to MeWo cells. Medium is removed from MeWo cells and replacedwith 1 mL of serum-free RPMI followed by the dropwise addition ofDNA/lipid complexes. Cells are incubated with complexes for 4-5 hours at37° C., 5% CO₂. Medium was removed and cells washed once with PBS. Thecells were then trypsinized and resuspended in 10% strip-serum RPMI(phenol red free). Cells were replated into an opaque 96-well tissueculture plate (Falcon, N.J.) at a cell density of 40,000 cells/well per100 μL media and assayed.

Plaque Assays

To determine whether the adenoviral constructs described above replicatepreferentially in melanocytes, plaque assays are performed. Plaquingefficiency is evaluated in the following cell types: melanoma cells(MeWo), prostate tumor cell lines (LNCaP), breast normal cell line(HBL-100), ovarian tumor cell line (OVCAR-3, SK-OV-3), and humanembryonic kidney cells (293). 293 cells serve as a positive control forplaquing efficiency, since this cell line expresses Ad5 E1A and E1Bproteins. For analyzing constructs comprising a melanocyte-specific TRE,cells that allow a melanocyte-specific TRE to function, such as the celllines provided above and cells that do not allow such function, such asHuH7, HeLa, PA-1, or G361, are used. The plaque assay is performed asfollows: Confluent cell monolayers are seeded in 6-well dishes eighteenhours before infection. The monolayers are infected with 10-fold serialdilutions of each virus. After infecting monolayers for four hours inserum-free media (MEM), the medium is removed and replaced with asolution of 0.75% low melting point agarose and tissue culture media.Plaques are scored two weeks after infection.

Example 19 In Vitro and In Vivo Assays of Anti-Tumor Activity

An especially useful objective in the development of urothelialcell-specific adenoviral vectors is to treat patients with bladdercancer. An initial indicator of the feasibility is to test the vector(s)for cytotoxic activity against cell lines and tumor xenografts grownsubcutaneously in Balb/c nu/nu mice.

In Vitro Characterization of CV876

Virus Yield Assay for CV876

5×10⁵ 293, RT-4, SW780, PA-1, G361, MKNI, HBL-100, Fibroblast (fromlung) and Smooth muscle cells (from bladder) were plated into each wellof six-well plates. Twenty-four hours later, medium was aspirated andreplaced with 1 ml of serum-free RPMI 1640 containing CV802 (wt.Ad5 withE3) or CV876 at a MOI of 2 pfu/cell. After a 4-h incubation at 37° C.,cells were washed with prewarmed PBS, and 2 ml of complete RPMI 1640were added to each well. After an additional 72 h at 37° C., the cellswere scraped into medium and lysed by three freeze-thaw cycles. Thelysates were tested for virus production by triplicate plaque assay for8-10 days under semisolid agarose on 293 cells.

Unlike wt. Ad5, CV802 which grows well in all of the cells tested, CV876replicates much better in permissive cells (293, RT-4 and SW780) than innon-permissive cells (PA-1, G361, MKN1, HBL-100 and primary cells) byabout 100-10000 fold. Noticeably, the replication in SW780 for CV876 isabout 100 fold less than CV802, which indicates the limitation of thisVirus in efficacy.

Growth Curve Experiment for CV876

5×10⁵ RT-4, PA-1, Smooth muscle and Fibroblast cells were plated intoeach well of six-well plates. Twenty-four hours later, medium wasaspirated and replaced with 1 ml of serum-free RPMI 1640 containingCV802 (wt.Ad5 with 133) or CV876 at a MOI of 2 pfu/cell. After a 4-hincubation at 37° C., cells were washed with prewarmed PBS, and 2 ml ofcomplete RPMI 1640 were added to each well. At different time points of0, 12, 24, 36, 48, 72, 96 and 120 h, the cells were scraped into mediumand lysed by three freeze-thaw cycles. The lysates were tested for virusproduction by triplicate plaque assay for 8-10 days under semisolidagarose on 293 cells.

Very similar as in virus yield assay, CV876 replicates well only in RT-4but not in primary cells and PA-1 over a 120 h period of time. However,CV876 does show a delay of replication in RT-4 compared to CV802.

Cytopathic Effect Assay for CV876

5×10⁵ 293, RT-4, SW780, PA-1, MKN1 and LNCap were plated into each wellof six-well plates. Twenty-four hours later, medium was aspirated andreplaced with 1 ml of serum-free RPMI 1640 containing CV802 (wt.Ad5 withE3) or CV876 at increasing MOI from 0.001 to 10 (the data shown was atMOI 1). After a 4-h incubation at 37° C., medium was replaced with 3 mlof complete RPMI 1640 and incubated at 37° C. for 6-8 days whencytopathic effect was observed for CV802 at MOI 0.01.

CV802 shows efficacy in all the cells tested while CV876 only kills thepermissive cells (293, RT-4 and SW780) but not the non-permissive cells(PA-1, MKN-1 and LNCap).

MTT Assay for CV876

2×10⁴ 293, RT-4, SW780, MKN1, PA-1, HBL-100, Smooth muscle cells (frombladder) and Fibroblast (from lung) were plated into each well of96-well plates. Twenty-four hours later, the cells were infected withCV802 and CV876 at increasing MOI from 0.001 to 10 in complete RPMI1640. A rapid colorimetric assay for cell growth and survival was run atdifferent time point of day 1, 3, 5, 7 and 10. The medium was replacedby 50 ul of MTT at 1 mg/ml solution, which is converted to an insolublepurple formazan by dehydrogenase enzymes present in active mitochondriaof live cells. After 3-4 h incubation at 37° C., the solution wasreplaced by isopropanol and the plates were incubated at 30° C. for 1 hand read at 560 nm test wavelength and 690 nm reference wavelength.

Similar as the results in CPE assay, CV876 shows efficacy only inpermissive cells but not in non-permissive cells. Again, in RT-4 andSW780, CV876 kills the cells much slower than CV802.

In Vitro Characterization of CV882

Virus Yield Assay for CV882

5×10⁵ 293, RT-4, SW780, G361, LNCap, HBL-100, MKN1, PA-1, Fibroblast andSmooth muscle cells were plated into each well of six-well plates.Twenty-four hours later, medium was aspirated and replaced with 1 ml ofserum-free RPMI 1640 containing CV802 (wt.Ad5 with E3) or CV882 at a MOIof 2 pfu/cell. After a 4-h incubation at 37° C., cells were washed withprewarmed PBS, and 2 ml of complete RPMI 1640 were added to each well.After an additional 72 h at 37° C., the cells were scraped into mediumand lysed by three freeze-thaw cycles. The lysates were tested for virusproduction by triplicate plaque assay for 8-10 days under semisolidagarose on 293 cells.

The replication of CV882 in permissive cells (293, RT-4 and SW780) iscomparable to CV802 (the difference is less than 100 fold) while itshows over 1000-1000000 fold difference in non-permissive cells (G361,LNCap, HBL-100, MKN1, PA-1 and primary cells).

Growth Curve Experiment for CV882

5×10⁵ RT-4, PA-1, and Fibroblast cells were plated into each well ofsix-well plates. Twenty-four hours later, medium was aspirated andreplaced with 1 ml of serum-free RPMI 1640 containing CV802 (wt.Ad5 withE3) or CV882 at a MOI of 2 pfu/cell. After a 4-h incubation at 37° C.,cells were washed with prewarmed PBS, and 2 ml of complete RPMI 1640were added to each well. At different time points of 0, 12, 24, 36, 48,72, 96 and 120 h, the cells were scraped into medium and lysed by three,freeze-thaw cycles. The lysates were tested for virus production bytriplicate plaque assay for 8-10 days under semisolid agarose on 293cells.

Very similar as in virus yield assay, CV882 replicates well only in RT-4but not in primary cells and PA-1 over a 120 h period of time.Additionally, CV882 shows better replication in RT-4 compared to CV876.

Cytopathic Effect Assay for CV882

5×10⁵ 293, RT-4, SW780, HBL-100, G361, PA-1 and Fibroblast cells wereplated into each well of six-well plates. Twenty-four hours later,medium was aspirated and replaced with 1 ml of serum-free RPNI 1640containing CV802 (wt.Ad5 with E3) or CV882 at increasing MOI from 0.001to 10 (the data shown was at MOI 1). After a 4-h incubation at 37° C.,medium was replaced with 3 ml of complete RPMI 1640 and incubated at 37°C. for 6-8 days when cytopathic effect was observed for CV802 at MOI0.01.

CV802 shows efficacy in all the cells tested while CV882 only kills thepermissive cells (293, RT-4 and SW780) but not the non-permissive cells(HBL-100, G361, PA-1 and Fibroblast cells).

MTT Assay for CV882

2×10⁴ RT-4, SW780, PA-1, HBL-100, U118 and Fibroblast were plated intoeach well of 96-well plates. Twenty-four hours later, the cells wereinfected with CV802 and CV882 at increasing MOI from 0.001 to 10 incomplete RPMI 1640. A rapid colorimetric assay for cell growth andsurvival was run at different time points of day 1, 3, 5, 7 and 10. Themedium was replaced by 50 ul of MTT at 1 mg/ml solution, which isconverted to an insoluble purple formazan by dehydrogenase enzymespresent in active mitochondria of live cells. After 3-4 h incubation at37° C., the solution was replaced by isopropanol and the plates wereincubated at 30° C. for 1 h and read at 560 nm test wavelength and 690nm reference wavelength.

Similar as the results in CPE assay, CV882 shows efficacy only inpermissive cells but not in non-permissive cells.

In Vitro Characterization of CV884

Virus Yield Assay for CV884

5×10⁵ 293, RT-4, SW780, G361, LNCap, HBL-100, MKN1, PA-1, Fibroblast andSmooth muscle cells were plated into each well of six-well plates.Twenty-four hours later, medium was aspirated and replaced with 1 ml ofserum-free RPMI 1640 containing CV802 (wt.Ad5 with E3) or CV984 at a MOIof 2 pfu/cell. After a 4-h incubation at 37° C., cells were washed withprewarmed PBS, and 2 ml of complete RPMI 1640 were added to each well.After an additional 72 h at 37° C., the cells were scraped into mediumand lysed by three freeze-thaw cycles. The lysates were tested for virusproduction by triplicate plaque assay for 8-10 days under semisolidagarose on 293 cells.

The replication of CV884 is very similar as CV802 in permissive cells(293, RT-4 and SW780) but shows over 1000 fold difference with CV802 innon-permissive cells (G361, LNCap, HBL-100, MKN1, PA-1 and primarycells). CV884 shows better efficacy than CV876 and CV882 without losingmuch specificity.

Growth Curve Experiment for CV884

5×10⁵ RT-4, PA-1, Smooth muscle and Fibroblast cells were plated intoeach well of six-well plates. Twenty-four hours later, medium wasaspirated and replaced with 1 ml of serum-free RPMI 1640 containingCV802 (wt.Ad5 with E3) or CV884 at a MOI of 2 pfu/cell. After a 4-hincubation at 37° C., cells were washed with prewarmed PBS, and 2 ml ofcomplete RPMI 1640 were added to each well. At different time points of0, 12, 24, 36, 48, 72, 96 and 120 h, the cells were scraped into mediumand lysed by three freeze-thaw cycles. The lysates were tested for virusproduction by triplicate plaque assay for 8-10 days under semisolidagarose on 293 cells.

Very similar as in virus yield assay, CV884 replicates very well only inRT-4 (similar as CV802) but not in primary cells and PA-1. Again, thereplication of CV884 is better than CV882 and CV876.

Cytopathic Effect Assay for CV884

5×10⁵ 293, RT-4, SW780, G361, PA-1 and Fibroblast cells were plated intoeach well of six-well plates. Twenty-four hours later, medium wasaspirated and replaced with 1 ml of serum-free RPMI 1640 containingCV802 (wt.Ad5 with E3) or CV884 at increasing MOI from 0.001 to 10 (thedata shown was at MOI 1). After a 4-h incubation at 37° C., medium wasreplaced with 3 ml of complete RPMI 1640 and incubated at 37° C. for 6-8days when cytopathic effect was observed for CV802 at MOI 0.01.

CV802 shows efficacy in all the cells tested while CV884 only kills thepermissive cells (293, RT-4 and SW780) but not the non-permissive cells(G361, PA-1 and Fibroblast cells).

MTT Assay for CV884

2×10⁴ 293, RT-4, SW780, U118, Fibroblast and Smooth muscle cells wereplated into each well of 96-well plates. Twenty-four hours later, thecells were infected with CV802 and CV884 at increasing MOI from 0.001 to10 in complete RPMI 1640. A rapid colorimetric assay for cell growth andsurvival was run at different time points of day 1, 3, 5, 7 and 10. Themedium was replaced by 50 ul of MTT at 1 mg/ml solution which isconverted to an insoluble purple formazan by dehydrogenase enzymespresent in active mitochondria of live cells. After 3-4 h incubation at37° C., the solution was replaced by isopropanol and the plates wereincubated at 30° C. for 1 h and read at 560 nm test wavelength and 690nm reference wavelength.

Similar as the results in CPE assay, CV884 shows strong efficacy(similar as wt. Ad5) only in permissive cells but not in non-permissivecells.

In Vivo Activity of CV808

Mice were given subcutaneous (SC) injections of 1×10⁶ sW780 cells. Whentumors grew to about 500 mm³, CV808 was introduced into the mice (5×10⁷PFU of virus in 0.1 ml PBS and 10% glycerol) intratumorally. Controlmice received vehicle alone. Tumor sizes were measured weekly. The dataindicate that CV808 was effective at suppressing tumor growth.

While it is highly possible that a therapeutic based on the virusesdescribed here would be given intralesionally (i.e., direct injection),it would also be desirable to determine if intravenous (IV)administration of adenovirus vector can affect tumor growth. If so, thenit is conceivable that the virus could be used to treat metastatic tumordeposits inaccessible to direct injection. For this experiment, groupsof mice bearing bladder epithelial tumors are inoculated with 10⁸ to10¹⁰ PFU of an adenoviral vector by tail vein injection, or with bufferused to carry the virus as a negative control. The effect of IVinjection of the adenoviral vector on tumor size is compared to vehicletreatment.

Example 20 Synergistic Effect of CV 890 with Chemotherapeutics

Materials and Methods

Cells

Hepatocellular carcinoma cell lines HepG2, Hep3B, PLC/PRF/5, SNU449, andSk-Hep-1, Chang liver cell (human normal liver cells), as well as othertumor cell lines PA-1 (ovarian carcinoma), UM-UC-3 (bladder carcinoma),SW 780 (bladder carcinoma), HBL100 (breast epithelia), Colo 201 (Colonadenocarcinoma), U 118 MG (glioblastoma) and LNCaP (prostate carcinoma)were obtained from the American Type Culture Collection. HuH-7 (livercarcinoma) was a generous gift of Dr. Patricia Marion (StanfordUniversity). 293 cells (human embryonic kidney containing the E1 regionof Adenovirus) were purchased from Microbix, Inc. (Toronto, Canada). Theprimary cells nBdSMC (normal human bladder smooth muscle cells), nHLFC(normal human lung fibroblast cells), and nHMEC (normal human mammaryepithelial cells) were purchased from Clonetics (San Diego, Calif.). Alltumor cell lines were maintained in RPMI 1640 (BioWhittaker, Inc.)supplemented with 10% fetal bovine serum (Irvine Scientific), 100 U/mlpenicillin and 100 ug/ml streptomycin. Primary cells were maintained inaccordance with vendor instructions (Clonetics, San Diego). Cells weretested for the expression of AFP by immunoassay (Genzyme Diagnostics,San Carlos, Calif.).

Virus Yield and One-Step Growth Curves

Six well dishes (Falcon) were seeded with 5×10⁵ cells per well of callsof interest 24 hrs prior to infection. Cells were infected at anmultiplicity of infection (MOI) of 2 PFU/cell for three hours inserum-free media. After 3 hours, the virus containing media was removed,monolayers were washed three times with PBS, and 4 ml of complete media(RPMI1640+10% FBS) was added to each well. 72 hours post infection,cells were scraped into the culture medium and lysed by three cycles offreeze-thaw.

The one-step growth curves time points were harvested at various timepoints after infection. Two independent infections of each viruscell-combination were titered in duplicate on 293 cells (Yu et al.,1999, Cancer Research, 59:1498-1504.

Northern Blot Analysis

Hep3B or HBL100 cells were infected at an MOI of 20 PFU/cell (plaqueforming unit per cell) with either CV802 or CV890 and harvested 24 hourspost infection. Total cell RNA was purified using the RNeasy protocol(Qiagen). The Northern blot was conducted using NorthernMax Plusreagents (Ambion, Austin, Tex.). 5 ug of RNA was fractionated on a 1%agarose, formaldehyde-based denaturing gel and transferred to aBrightStar-Plus (Ambion) positively charged membrane by capillarytransfer. The antisense RNA probes for E1A (adenovirus genome 501 bp to1141 bp) or E1B (1540 bp-3910 bp) were PCR products cloned in pGEM-Teasy (Promega) and transcription labeled with [α ³²P] UTP. Blots werehybridized at 68° C. for 14 hours with ZipHyb solution and washed usingstandard methods (Ambion). Membranes were exposed to BioMax film(Kodak).

Western Blot Analysis

Hep3B or HBL100 cells were infected at MOI of 20 PFU/cell with eitherCV802 or CV890 and harvested 24 hours post infection. Cells were washedwith cold PBS and lysed for 30 min on ice in (50 mM Tris, pH8.0, 150 mMNaCl, 1% IGEPAL CA360 a NP40 equivalent (Sigma), 0.5% sodiumdeoxycholate, and protease inhibitor cocktail from (Roche, Palo Alto,Calif.). After 30 min centrifugation at 4 C, the supernatant washarvested and the protein concentration determined with protein assayESL kit (Roche). Fifty micrograms of protein per lane were separated on816% SDS-PAGE and electroblotted onto Hybond ECL membrane (AmershamPharmacia, Piscataway, N.J.). The membrane was blocked overnight in PBST(PBS with 0.1% Tween-20) supplemented with 5% nonfat dry milk. Primaryantibody incubation was done at room temperature for 2-3 hrs withPBST/1% milk diluted antibody, followed by wash and 1 hr incubation withdiluted horseradish peroxidase-conjugated secondary antibody (Santa CruzBiotechnology Inc., Santa Cruz, Calif.). Enhanced chemiluminescence(ECL; Amersham Pharmacia) was used for the detection. E1A antibody(clone M58) was from NeoMarkers (Fremont, Calif.), E1B-21 kD antibodywas from Oncogene (Cambridge, Mass.). All antibodies were used accordingmanufacturer's instruction.

Cell Viability Assay and Statistical Analysis

To determine the cell killing effect of virus and chemotherapeutic agentin combination treatment, a cell viability assay was conducted aspreviously described with modifications (Denizot, 1986, JournalImmunology. Methods, 89:271-277). On 96 well plates, cells of interestwere seeded at 10,000 calls per well 48 hr prior to infection. Cellswere then treated with virus alone, drug alone, or in combination. Cellviability was measured at different time points by removing the media,adding 50 μl of 1 mg/ml solution of MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)(Sigma, St. Louis, Mo.) and incubating for 3 hrs at 37° C. Afterremoving the MTT solution, the crystals remaining in the wells weresolubilized by the addition of 50 μl of isopropanol followed by 30 Cincubation for 0.5 hr. The absorbency was determined on a microplatereader (Molecular Dynamics) at 560 nm (test wavelength) and 690 nm(reference wavelength). The percentage of surviving cells was estimatedby dividing the OD₅₅₀-OD₆₅₀ of virus or drug treated cells by theOD₅₅₀-OD₆₅₀ of control cells. 6 replica samples were taken for each timepoint and each experiment was repeated at least three times.

For statistical analysis, CurveExpert (shareware by Daniel Hyams,version 1.34) was used to plot the dose-response curves for virus anddrugs. Based upon the dose-response curves, the isobolograms were madeaccording to the original theory of Steel and Peckham (1993, Int. J.Rad. One. Biol. Phys., 5:85) and method described in Aoe et al. (1999,Anticancer Res. 19:291-299).

Animal Studies

Six to eight week old athymic BALB/C nu/nu mice were obtained fromSimonson Laboratories (Gilroy, Calif.) and acclimated to laboratoryconditions one week prior to tumor implantation. Xenografts wereestablished by injecting 1×10⁶ Hep3B, HepG2 or LNCaP cells suspended in100 μl of RPMI 1640 media subcutaneously. When tumors reached between200 mm³ and 300 mm³, mice were randomized and dosed with 100 μl of testarticle via intratumoral or the tail vein injection. Tumors weremeasured in two dimensions by external caliper and volume was estimatedby the formula [length (mm)×width (mm)²]/2. Animals were humanely killedwhen their tumor burden became excessive. Serum was harvested weekly byretro-orbital bleed. The level of AFP in the serum was determined by AFPImmunoassay kit (Genzyme Diagnostics, San Carlos, Calif.). Thedifference in mean tumor volume and mean serum AFP concentration betweentreatment groups was compared for statistical significance using theunpaired, two-tailed, t-test.

Transcription and Translation of E1A/E1B Bicistronic Cassette of CV890in Different Cells

In wild type adenovirus infection, E1A and E1B genes produce a family ofalternatively spliced products. It has been found that there are fiveE1A mRNAs, among them 12S (880 nucleotides, nts) and 13S (1018 nts)mRNAs are the dominant ones that are expressed both early and late afterinfection. The 12S and 13S mRNAs encode the gene product of 243 aminoacids (243R) and 289 amino acids (289R) respectively (reviewed by Shenk,1996). The two major E1B transcripts that code for 19 kD and 55 kDproteins are 12S (1031 nts) and 22S (2287 nts) mRNAs. E1B 12S mRNA onlycodes the 19 kD product, whereas the 22S mRNA codes for both 19 kD and55 kD products due to different initiation sites during translation. Inthe current study, the generation of E1A-IRES-E1B bicistronic cassettewas expected to change the pattern of E1A and E1B transcripts in viralinfection. Therefore, Northern blot analysis was conducted to evaluatethe steady-state level of E1A and E1B transcripts. First, CV802 or CV890were infected to Hep3B (AFP) or HBL100 (AFP) cells for 24 hours. Thetotal RNA samples were separated on agarose gels and processed forNorthern blot by hybridizing to antisense RNA probes. The Northern blotwith E1A probe visualized the 12S and 13S mRNAs in both wild type CV802infected cells. For CV890, E1A transcripts can only be seen in Hep3Bcells, indicating the conditional transcription of E1A. It is ofinterest to find that in CV890, there is only one large transcript(about 3.51 Kb), whereas the 12S and 13S mRNAs are no longer present.This large transcript indicates the continuous transcription ofE1A-IRES-E1B bicistronic cassette, suggesting an alteration of viral E1Asplicing pattern in CV890. Transcription of E1B from CV890 also appearsto be AFP-dependent. It is clear that both 12S and 22S mRNAs of E1B werepresent in wild type CV802 samples, whereas the 128 mRNA and an enlarged22S mRNA (3.5 Kb) appeared in CV890 infected cells. Obviously, theidentity of this enlarged mRNA is the same 3.5 Kb transcript asvisualized in E1A blot, which is from the transcription of E1A/E1Bbicistronic cassette. Therefore, the E1B mRNA is tagged after E1A mRNAin this large transcript. This large transcript contains all the codinginformation for E1A, E1B 19 kD and E1B 55 kD. The mRNA splice patternthat appears in CV802 is not valid in CV890, the 12S mRNA with E1B probedisappeared. Meanwhile, in the E1B Northern blot, due to the selectionof our E1B probe (1540 bp-3910 bp), mRNA of the Adenovirus gene IX (3580bp-4070 bp), the hexon-associated protein, was also detected. In CV890infected Hep3B cells, gene IX expression is equivalent to that of CV802,whereas in CV890 infected HBL100, its expression was also completelyshut down. This result further demonstrated that the AFP controlledE1A/E1B expression is the key for late gene expression as well as viralreplication.

Results of the same samples in the Western blot also indicate that CV890has AFP dependent expression of E1A and E1B. Under our experimentalconditions, E1A expression level of CV890 in Hep3B cells is similar tothat of CV802. However, when E1B 19 kD protein was detected, it wasfound that the expression level was much lower than CV802 E1A.Previously, it has been addressed that IRES-mediated second gene hasless expression (Mizuguchi et al., 2000, Mol. Ther. 1:376-382). Takentogether, CV890 infection in permissive Hep3B cells can produce normalamounts of E1A and lesser amounts of E1B proteins capable of initiatinga normal productive infection. In AFP⁻ cells, however, this process wasattenuated due to a lack of E1A and E1B gene transcription andtranslation. These data demonstrated that the expression of both E1A andE1B genes are under the control of AFP TRE and the artificial E1A/E1Bbicistronic cassette is functioning properly in CV890.

In Vitro Replication Specificity of CV890 in Tumor Cells and PrimaryCells

From in vitro comparison of virus yield, CV890 has a better specificityprofile than CV732 (CV732 is an AFP-producing, cell-specific adenovirusvariant in which the E1A gene is under control of AFP-TRE). In order togain further insights of using CV890 in liver cancer therapy, more tumorcell lines and primary cells were tested to characterize in vitro virusreplication. First, all cells in the study were analyzed for their AFPstatus by AFP immune assay. Based on AFP produced in the cells andmedia, all the cells were divided into three groups, high (>2.5 μg/10⁶cells/10 days), low (<0.6 μg/10⁶ cells/10 days) and none (undetectablein our study) (Table 15). It was confirmed that replication of CV890 indifferent cell lines correlates well with the AFP status of the hostcell. Among the group of liver cell lines, CV890 only replicates well inAFP⁺ cells, including Hep3B, HepG2, Huh7, SNU449 and PLC/PRF/5. Theamount of AFP required for the promoter activity seems very low as oneof the hepatoma cell lines, SNU449, a previous reported AFP⁻ cell (Parket al., 1995, Int. J. Cancer 62:276-282), produces very low AFP (about60 ng/10⁶ cells/10 days) compared to other cells. Nevertheless, evenwith very low amount of AFP, SNU449 cells can still support CV890replication to the extent comparable to cells producing significantlyhigher levels of AFP such as HepG2. Compared to CV802, CV890 isattenuated 5,000 to 100,000 fold in cells that do not produce AFP,including the hepatoma cell Sk-Hep1 and Chang liver cell, other tumorcells and primary cells. Taken together the results indicate that CV890has shown a good specificity profile from a broad spectrum of tumorcells. Among them, only the AFP⁺ liver cells, AFP production level fromhigh to low, are permissive for CV890.

In another experiment, CV890 was compared to CV802 for their single stepgrowth curves on different cells. Results demonstrated that CV890 has asimilar growth kinetics to wild-type CV802 in AFP⁺ cells except thatvirus yields are slightly lower (2-8 fold) in low AFP producing cells.In consideration of experimental error, there is no dramatic differencein the replication of CV890 and CV802 in AFP⁺ hepatoma cells. However,the growth curves of CV890 in AFP-cells showed clear attenuation. Duringa 5 day experiment, CV890 failed to replicate in AFP⁻ cells includinghepatoma cell (Change liver) and primary cells (nHLFC). From all the invitro virus replication studies, it is clear that replication of CV890is under the tight control of AFP-TRE and this adenovirus variant has anexcellent specificity profile of preferentially targeting AFP producinghepatocellular carcinoma cells.

In Vivo Specificity and Efficacy of CV890

CV890 specificity was also evaluated in animals bearing prostate cancerLNCaP xenografts. In this in vivo test, nude mice with prostatexenograft were intravenously injected with either CV890 or CV787, aprostate cancer specific adenovirus variant (Yu et al., 1999, CancerResearch, 59:42004203). Tumor volumes were documented and indicated thatonly CV787 had a significant antitumor efficacy in LNCaP xenografts,while tumors in the animals treated with CV890 grew, from 400 mm³ toapproximately 1200 mm³ in six weeks, similar to the group treated withvehicle. This study indicates that CV890 does not attack LNCaP xenograftand keeps the good specificity profile under in vivo conditions.

To evaluate in vivo antitumor efficacy of CV890, different studies werecarried out in the nude mouse model harboring human hepatoma xenografts.First, BALB/c nu/nu mice with HepG2 or Hep3B xenografts wereestablished, animals were further challenged with single dose ormultiple doses of CV890 into the tumor mass (intratumoraladministration, IT) or via their tail vein (intravenous administration,IV). Tumor volume and the level of serum AFP were monitored weekly afterthe start of treatment, and hence the efficacy of the treatment wasdetermined. The in vitro cytotoxicity study has demonstrated that CV890has a better cytolytic effect than CV732. In order to further examinetheir antitumor activity, we first conducted animal study to compareCV890 to CV732. Animals harboring 300 mm³ Hep3B xenograft were grouped(n=6) and injected with vehicle alone (control group), CV890 (1×10¹¹particles/dose, CV890 group), or CV732 (1×10¹ particles/dose, CV732group). The Hep3B xenograft is a very aggressive tumor model and tumorsgrow very fast. Most animals can not survive long because of excessivetumor burden. During a six week study, single intravenous administrationof CV890 have shown significant tumor growth inhibition, whereas controlmice had over 10 fold tumor growth at week 5. In the group treated withCV732, single dose IV injection also reduced the tumor growth ascompared to control group, however, it was much less effective comparedto CV890. For example, the average tumor volume of the CV890 treatedgroup dropped from 312 mm³ to 219 mm³, while tumor volume increased from308 mm³ to 1542 mm³ 5 weeks after treatment in control. Both controlgroup and the CV732 group were terminated at week 5 because excessivetumor size. Previously, CV732 has been demonstrated to restrict thehepatoma tumor from growth after 5 doses of intravenous administration.Similar efficacy can be achieved with just a single intravenousadministration of CV890, indicating that under in vivo conditions, CV890has better efficacy than CV732 in hepatoma xenografts. In thisexperiment, 4 out of five CV890 treated mice were tumor free three weeksafter treatment. However, in CV732 group, xenografts in two mice stoppedgrowing but none of treated animals were tumor free through the six-weekexperiment. There was no tumor reduction in this group or the controlgroup of animals. By statistical analysis, the differences in meanrelative tumor volumes and serum AFP concentrations between CV890treated and CV732 treated or vehicle treated tumors are significant(p<0.01)). Taken together, these studies suggest that CV890 has asignificant antitumor activity and its oncolytic efficacy is better thanCV732, an adenovirus variant similar to AvE1a04I, in which the AFP TREwas applied to control E1A alone (Hallenback et al, 1999, Hum. Gene.Ther., 10:1721-1733).

Synergistic Antitumor Efficacy of CV890 in Combination withChemotherapeutic Agents

In this example, different chemotherapeutic agents were tested incombination with CV890 for their in vitro killing effect in Hep3B orHepG2 cells. Drug concentrations were optimized to the extent that theywould not generate extensive cytotoxic effect on their own. Under suchconditions, some agents had shown higher cell killing effect incombination with CV890. Among them, doxorubicin, a drug currently usedin treatment of HCC showed synergistic cytotoxicity with CV890. Inexperiments using doxorubicin together with CV890 on Hep3B cells,doxorubicin at 10 ng/ml did not generate cytotoxicity, whereas CV890 atan MOI of 0.01 (pfu/cell) only had about 35% of cell killed at day 9.However, when both were applied together, 90% cells were killed 9 daysafter treatment. In order to determine the potential synergistic effectfrom the combination treatment, the MTT cell viability data weresubjected to further statistical analysis. FIG. 38 shows arepresentative IC₅₀ isobologram of doxorubicin and CV890 on Hep3B cellsat day 5. First, the dose-response curves of doxorubicin alone or CV890alone were made. Based on the original theory of Steel and Peckham(1993) and method by Aoe et al. (1999), three isoeffect curves (mode Iand mode 2a, 2b) were constructed. From this isobologram, several datapoints were in the synergy or additive area, indicating that combinationof CV890 and doxorubicin provides synergistic effect on killing of Hep3Bcells.

Although CV890 alone has good antitumor activity, we applied combinationtherapy with doxorubicin for in vivo evaluation of synergy. Animalsharboring 300 mm³ Hep3B xenografts were grouped (n=6) and injected withvehicle alone (control group), CV890 alone (1×10¹¹ particles/dose, CV890group), doxorubicin alone (10 mg/kg, doxorubicin group), or CV890 incombination with doxorubicin (combination group). FIG. 38 shows weeklychange of the relative tumor size normalized to 100% at day 1. In thisexperiment, by week six, all animals in the control group had excessivetumor which has increased by 700% of baseline, whereas in CV890 groupand combination group, animals had either tumor free or tumor reduction.Of the eight Hep3B xenografts, treated with CV890, three animals (37.5%)had no palpable tumor at week 5, another three animals had tumorregressed by more than 60%. In combination group, four out of eightanimals were tumor free from week 5, another four animals had tumorreduction about 90%. All the animals in the CV890 and combination groupwere alive and tumor was suppressed even ten weeks following treatmentwhereas the control animals were sacrificed for excessive tumor burdenafter week 6. Furthermore, CV890 also caused a drop in the serum AFPconcentration in these mice. Statistical analysis shows that differencesin mean relative tumor volumes and serum AFP concentrations betweenCV890 and vehicle treated group or combination and doxorubicin treatedgroup are significant at different times (p<0.005).

The strong efficacy in the combination treatment shows that single IVinjection of CV890 in combination of doxorubicin can eradicateaggressive Hep3B xenografts in most of the animals. TABLE 15 AFPproduction in different tumor cells AFP CELLS (ng/10⁶cells/10days) Hep3B2645 High HepG2 3140 HuH7 4585 SNU449 60 Low PLC/PRF/5 600 Chang 0 NoneSK-Hep1 0 HBL100 0 PA-1 0 LoVo 0

Example 21 CV706 in Combination with Irradiation Produces Synergy

Materials and Methods

Cell Culture and Virus

The human LNCaP (prostate carcinoma), OVCAR-3 (ovary carcinoma) andHBL-100 (breast epithelia) cell lines were obtained from the AmericanType Culture Collection (ATCC, Rockville, Md.). The human embryonickidney cell line, 293, which expresses the Adenoviral E1A and E1B geneproducts, was purchased from Microbix Biosystem, Inc. (Toronto, Canada).Cells were maintained at 37° C. with 5% CO₂ in RPMI 1640 supplementedwith 10% fetal bovine serum (FBS, Hyclone, Utah), 100 units/mlpenicillin and 100 μg/ml of streptomycin (Life Technologies,Gaithersburg, Md.).

CV706 is a prostate-specific replication competent Adenovirus variant.One prostate-specific transcription response element (TRE), the humanprostate-specific antigen promoter and enhancer (PSE), was insertedupstream of the E1A encoding region in the viral genome (Rodriguez etal., 1997, Cancer Research, 57: 2559-2563). Similarly, CV787 is also aprostate-specific replication competent Adenovirus variant, whichcontain two prostate-specific TREs, the probasin promoter and PSE,inserted upstream of the E1A and E1B encoding regions in the viralgenome, respectively (Yu et al., 1999, supra). Both CV706 and CV787 arecurrently in clinical trials for organ-confined prostate cancer andmetastatic hormone refractory prostate cancer (DeWeese et al., 2001).

Cell Viability and Irradiation

MTT assays were performed to measure cell viability as described by (Yuet al, 1999, supra). Briefly, HBL-100, OVCAR-3 and LNCaP cells (2×10⁴cells/well, 96 well plate) were either infected with CV706 or CV787 atvarious MOI (from 0.0001 to 1) or treated with irradiation at theindicated dosages. Cells were incubated in growth medium for 24 hr toallow for viral replication. After 24 hr, cells were exposed to a singledose of γ-irradiation (0˜40 Gy) (Mark 1 Research Irradiator Model#1608A, Caesium 137 source). Cell viability was measured at the timesindicated by removing the media and replacing it with 50 μl of 1 mg/mlsolution of MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)(Sigma, St. Louis, Mo.) and incubating for 3 hrs at 37° C. Afterremoving the MIT solution, the crystals remaining in the wells weresolubilized by the addition of 50 μl of isopropanol and placed in a 30°C. incubator for 30 min for the crystals to dissolve. Plates werevibrated for 10 sec prior to reading. The absorbency was determined on amicroplate reader (Molecular Dynamics) at 560 nm (test wavelength) and690 nm (reference wavelength). At least, 8 replica samples were takenfor each time point and the percentage of surviving cells was estimatedby dividing the OD₅₆₀-OD₆₉₀ of virus infected cells by the OD₅₆₀-OD₆₉₀of mock infected cells.

Statistical Analysis

The dose-response interactions between CV706 and irradiation at thepoint of IC₅₀ were evaluated by the isobologram method of Steel andPeckham as modified by Aoe et al. (Aoe et al. 1999, Anticancer Res.19:291-299). The IC₅₀ defined as the concentration of drug that produced50% cell growth inhibition, i.e. 50% reduction in absorbance.Isobolograms (three isoeffect curves, model 1 and model 2) were computedas described previously (Yu et al., 2001). Fractional tumor volume (FTV)relative to untreated controls was determined based on the methoddescribed previously (Yokoyama et al., 2000; Yu et al., 2001, CancerResearch).

On Step Growth Curve and Virus Yield

One-step growth curve of CV706 in the presence and absence ofirradiation were performed in LNCaP cells to determine burst size.Monolayers of LNCaP cells were infected with CV706 at MOIs 0.01, 0.1and 1. After 24 hour incubation at 37° C. with 5% CO₂, cells wereexposed to a single dose of γ-irradiation at 10 Gy. At the indicatedtimes thereafter, duplicate cell samples were harvested and lysed bythree cycles of freeze-thawing. Virus yield was determined by plaqueassay as described in (Yu et al., 1999, Cancer Research, 59:1498-1504).

In Vivo Antitumor Efficacy

Six to eight week old athymic Balb/c nu/nu mice were obtained fromSimonson Laboratories (Gilroy, Calif.) and acclimatized to laboratoryconditions one week prior to tumor implantation. Xenografts wereestablished either by injecting 1×10⁶ LNCaP cells subcutaneously nearthe small of the back suspended in 100 μl of RPMI 1640 and 100 μl ofmatrigel (back tumor) or by injecting cells into the right gastrocnemiusmuscle (i.m.) (leg tumor). When tumors reached between 300 mm³ and 500mm³, mice were randomized into groups of four. The first group receivedCV706 at day 0 via intratumoral (i.t.) administration. CV706 was dilutedby PBS containing 10% glycerol and injected into tumor as 0.4 μl ofdiluted virus (1×10⁷ particles) per mm³ of tumor using a 28-gaugeneedle. The second group was given irradiation only. For irradiationmice were immobilized in lucite chambers and their whole body wasshielded with lead except for the tumor bearing sites on their back ortumor-bearing hind leg. This tumor-bearing site in back or leg wasirradiated with a Mark 1 Research Irradiator (Model #1 608A, J.H.Shepherd Associates) at various doses (0, 5, 10 and 20 Gy) 1 day afterCV706 injection or mock injection. The third group was given CV706(i.t.) at day 0 and irradiated at the same doses at day 1. As a control,a fourth group was treated with virus dilution buffer (i.e. control)i.t. at day 0. Tumors were measured weekly in two dimensions by externalcaliper and volume for back tumors was estimated by the formula [length(mm)×width (mm)^(2])/2 (Yu et al., 1999b). Volumes of i.m. leg tumorswere determined using the following formula (Alfieri and Hahn, 1978,Cancer Research, 38:3006-3011): volume (cm³) d′3−(0.6)²d′, where d′ isthe average diameter of the tumor-bearing leg (cm), and the product(0.6)²d′ is the correction factor for normal leg volume. Animals werehumanely killed when their tumor burden became excessive. The differencein relative tumor volumes between treatment groups was compared forstatistical significance using the type 2 (two-sample equal variance),two-tailed, t-test. Blood samples were collected at various time pointsby retro-orbital bleed for determining prostate-specific antigen.Federal and institutional guidelines for animal care were followed.

Histochemistry Analysis

Four groups of mice (n=6) were treated with vehicle, CV706 (1×10⁷particles per mm³ of tumor), irradiation (10 Gy) or a combination ofCV706 and irradiation. Half the animals were sacrificed on day 7 and theother half on day 14. The tumor samples were embedded in paraffin blocksand 4-μm sections were cut and stained with Hematoxylin and Eosin (H&E).Histology methods for detecting Adenovirus antigens were as described(Yu et al., 1999, Cancer Research, 59:4200-4203). The necrotic cellswere scored on coded slides at light microscopy at ×400 magnification.The number of necrosis was based on scoring 500 points per section aseither necrotic or normecrotic. The average necrosis score wascalculated based on counting in 10 fields distributed evenly across thearea of tumor section. The light-microscopic features used to identifynecrosis included cell size, indistinct cell border, eosinophiliccytoplasm, loss or condensation of the nucleus, and associatedinflammation (Milross et al., 2000). To assess the effect of CV706,irradiation or the combination treatment on tumor vascularization, thenumber of blood vessels was counted at a magnification of ×400 and theaverage blood vessels were calculated from 10 fields distributed evenlyacross the area of whole tumor section. Apoptotic cells were detectedusing TUNEL assay (Roche Molecular Biochemicals, Indianapolis, Ind.) assuggested by the manufacturer. The morphological features used toidentify apoptosis in the tumor sections have been previously described,associated with positive terminal deoxynucleotidyl transferase-mediatednick end labeling staining (Milross, et al., 2000). The apoptotic cellswere scored on coded slides at ×400 magnification and average score ofapoptotic cells was calculated from 10 fields of normecrotic areasselected randomly across each tumor section.

Results

CV706 in Combination with Irradiation Produce Synergistic Cytotoxicityin Prostate Carcinoma LNCaP Cells

To study the potential interaction between a prostate-specificAdenovirus variant CV706 and radiation in vitro, the effectiveness ofcombined treatment of several combinations of CV706 and irradiation atvarious doses was evaluated in the PSA-producing prostate carcinomaLNCaP cell line. LNCaP cells were either mock-infected, or infected withCV706. One day later, cells received a single dose of γ-irradiation (0,5 Gy, 10 Gy and 20 Gy) and the cell viability was then determined atvarious time points by the MTT assay. Several viral MOIs and radiationdoses were tested to determine the dose-response curves in LNCaP cells,such that the selected dose shows greater combined efficacy withradiation or virus, but minimal cell killing when treated with the samedose of virus alone or radiation alone. Infecting LNCaP cells with CV706at an MOI of 0.01 resulting in 80% cell survival 5 days after infection,while irradiation at a dose of 10 Gy resulted in 78% survival 5 daysafter treatment. However, when CV787 and radiation were combined atthese doses, cell survival dropped to 20% 5 days after treatment. Cellviability dropped further to 8% 9 days after combination treatment,while cells treated with virus at MOI 0.01 alone or radiation 10 Gyalone retained 70% or 60% cell viability, respectively.

Isobolograms were generated from the models to determine the presence ofsynergy, additivity, or antagonism between CV706 and irradiation. Theresults indicate that sequential exposure to CV706 followed byirradiation produced synergistic cytotoxicity. The enhanced cytotoxicitywas also observed in LNCaP cells when CV787, a second prostate-specificAdenovirus variant, was combined with radiation Taken together, our invitro data demonstrate that prostate-specific Adenovirus variants incombination with irradiation produce synergistic cell cytotoxicity inprostate carcinoma LNCaP cells.

Irradiation Increases CV706 Burst Size in LNCaP Cells

Irradiation kills mammalian cells in the reproductive (also known asclonogenic) death pathway. DNA is the target, and double-stranded breaksin the DNA are regarded as the specific lesions that initiate thislethal response. Most radiation induced DNA double-stranded breaks arerapidly repaired by constitutively expressed DNA repair mechanisms.Residual unrepaired or misrepaired breaks lead to genetic instabilityand to increased frequency of mutations and chromosomal aberrations(Garzotto et al., 1999). Because of its small target size, theadenoviral genome (36 kb) is far less likely to sustainradiation-induced damage as it is 10⁵-fold smaller than that of humancells (3×10⁶ kb).

To examine the effect of irradiation on virus replication, we performeda one-step growth curve. LNCaP cells were infected with CV706 at an MOIof 0.1 for 24 hrs, followed by irradiation at a dose of 10 Gy. Cellswere harvested at various times post-infection and the number ofinfectious virus particles was determined on 293 cells by standardplaque assay (Yu et al., 1999, supra). Although the initial rate ofincrease of CV706 in cells treated with CV706 and irradiation wassimilar to that of cells treated with CV706 alone, cells treated withCV706 and irradiation produced a larger burst size than CV706 alone. Forexample, cells treated with CV706 and irradiation produced 8,000 PFU percell 9 days post-infection, while the cells infected with CV706 alonegenerated about 500 PFU per cell 9 days after virus infection. A biggervirus burst size was also observed in the combination treatment ofirradiation and CV706 at MOIs 0.01 or 1. Cells treated with CV706 at MOIof 0.01, and 1 produced 15 and 3500 PFU per cell, whereas cells treatedwith CV706 at MOI of 0.01 and 1 combined with irradiation, produced4750, and 8700 PFU per cell respectively, at 9 days after virusinfection. Thus, irradiation does not inhibit CV706 replication, butsignificantly increases virus propagation.

Cytotoxicity of CV706 in Combination with Irradiation Remains to beSpecific to Prostate Cancer Cells

In order to evaluate whether the addition of radiation could change thespecificity of CV706's cytotoxic activity, we assess the specificity ofthe combination treatment of CV706 and radiation by measuring viabilityof various infected cell lines using the MTT assay. LNCaP, HBL-100 andOVCAR-3 cells were infected with CV706 at an MOI of 0.01 for 24 hrs,followed by a single dose of radiation at 10 Gy. The percentage of cellviability versus time post treatment was plotted. The combination ofCV706 and radiation was toxic to LNCaP cells, but not to HBL-100 andOVCAR-3 cells. There were few surviving LNCaP cells 9 days afterinfection. In contrast, the viability of HBL-100 and OVCAR-3 cellstreated with CV706 and radiation was more than 90% throughout the courseof the experiment, similar to that of cells treated with radiationalone. This data suggests that combination with irradiation does notalter CV706's specificity.

Synergistic Efficacy of CV706 in Combination with Irradiation In Vivo

The in vivo antitumor efficacy of CV706 in combination with irradiationwas assessed in the LNCaP mouse xenograft model. We have shownpreviously that a single intratumoral administration of CV706 at 5×10⁸particles per mm³ of tumor can eliminate subcutaneous xenograft tumorsin 6 weeks (Rodriguez et al., 1997, supra) Established human prostatecancer xenografts (LNCaP cells) were treated with either vehicle, CV706(1×10⁷ particles/mm³), irradiation (10 Gy), or both CV706 andirradiation. For the combination treatment, animals were intratumorallyinjected with either CV706 or vehicle, and 24 hours later, animalsreceived a single dose of irradiation. In this study, a single dose of10Gy was used because it caused a tumor growth delay in a previous pilotstudy. The dose of 1×10⁷ particles per mm³ of tumor was selected basedon our previous studies on its antitumor efficacy (Yu et al., 1999,supra.

The tumor volume data shows that there was a significant decrease intumor volume between control and all treatment groups. In all casesalthough single doses of CV706 or irradiation were effective inproducing tumor growth inhibition, the combination of the two showedsignificant tumor regression. For example, tumor volume of the grouptreated with irradiation (10 Gy) was 119.76% of baseline 6 weeks aftertreatment, while the tumor volume of the group treated with CV706 was97.39% of baseline 6 weeks after administration. However, when CV706 wascombined with irradiation at similar doses, a statistically significantdrop in the relative tumor volume (4% of baseline) was observed(p<0.01). Additionally, relative PSA level in serum of mice was alsomonitored for anti-tumor efficacy. Relative PSA level in mice increasedto 370% of baseline 6 weeks after receiving vehicle treatment, increasedto 139% after receiving irradiation alone, reduced to 84% of baselineafter being treated with CV706 alone, whereas the PSA levels in micetreated with CV706 and irradiation decreased to less than 1% of theirstarting values within 6 weeks.

After 7 days, combination treatment showed more than additive effect ontumor growth inhibition at all the time points studied. On day 21, therewas more than 2-fold improvement in anti-tumor activity in thecombination group when compared with the expected additive effect. Atthis time point, both CV706 and irradiation (10 Gy) per se inhibitedtumor growth by 26% and 34%, respectively (fractional tumor volume,0.7419 mm³ and 0.6645 mm³, respectively) when compared with the controlgroup. This anti-tumor activity further improved with time. On day 42,the group treated with the combination of CV706 and irradiation showed a6.69-fold higher inhibition of tumor growth over the expected fractionaltumor volume. These observation further strengthen the idea of synergybetween CV706 and irradiation in the eradication of LNCaP xenografts.

Enhanced antitumor efficacy was also observed in the animal model inwhich the prostate cancer tumors are implanted in hind limb of mice. Inthis study, tumors were produced by inoculation of 1×10⁶ cells into limbmuscle. Those tumors which were attained a volume of 200 mm³ to 300 mm³were randomized into four groups and treated as described above for backtumors. As before the weekly tumor volume measurements showed thatcombination treatment of CV706 and irradiation led to significantantitumor activity in comparison to either CV706 or irradiation. Forexample, tumor volume of the group treated with irradiation (20 Gy) was70% of baseline 4 weeks after treatment, while the tumor volume of thegroup treated with CV706 (5×10⁷ particle per mm³ of tumor) was 75% ofbaseline 4 weeks after administration. However, when CV706 was combinedwith irradiation at these dose levels, the tumor volume dropped to 8% ofbaseline.

A series of experiments were then designed to examine the effects ofvarious factors, including the sequencing of the agents, timing ofirradiation following virus administration and irradiationfractionation. The effect of order of administration for the testedagents was examined in an in vivo study using back tumor xenograftmodel. LNCaP xenografts were irradiated 24 hr before or after CV706administration. Weekly measured tumor volume indicated that treatmentwith CV706 prior to irradiation was significantly superior toirradiation followed by CV706.

The second study was designed to evaluate the timing of irradiationfollowing virus administration. Tumors were treated with CV706 at day 0and followed by irradiation at various periods of time. The results ofaverage tumor volume indicated that similar antitumor efficacy wasachieved when tumors treated with CV706 at day 0 following byirradiation 1 day or 4 days after virus administration, both eliminatedtumors within 6 weeks after treatment. However, the antitumor activitywas decreased when the tumors were treated with irradiation 7 days afterCV706 administration.

The third study was designed to assess the effect of radiationfractionation on antitumor efficacy. Animals with human prostate cancertumors on their backs were randomized into five groups. Two of whichwere treated with either CV706 at day 0 followed by a single dose ofradiation at 10 Gy on day 1, or CV706 at day 0 followed with fourfractional doses of radiation at 2.5 Gy on day 1, 2, 6 and 8. Weeklymeasured tumor volume data indicated that both treatments eliminated thepre-existing tumors 6 weeks after treatment and produced an synergisticantitumor activity when compared to either agent alone. However, nosignificant difference in antitumor efficacy was observed between thesetwo combination groups as long as the total doses of irradiation was thesame.

Synergistic antitumor efficacy of CV706 in combination irradiation wasfurther documented by tumor histological analysis. First of all, morenecrotic cells were observed in the tumors treated with CV706 plusirradiation compared with either agent alone. The amount of necrosis intumors treated with CV706 alone was higher than control tumor or tumortreated with radiation. Evidence of necrosis and multifocal inflammationwas observed in a small portion of tumors treated with radiation. In thetumor treated with both the virus and radiation, a few virus-infectedcells were detected. Most of the cells in the sections were empty andvirtually devoid of cellular content. Significantly increases in theextent of necrosis was a dominant histological feature, which makes upabout 95% of the tumor mass in this treatment group. The averagenecrosis scores in a ×400 magnification for the tumors treated withvehicle, radiation, CV706 and both were 5.4±2.17, 67±48.24, 258.2±80.76and 461.6±37.87, respectively. The presence of mass necrosis in thetumors treated with CV706 or CV706 plus radiation suggests that theinduction of necrosis greatly attributes CV706 or CV706 plus radiation'santi-tumor efficacy in vivo. Student T test showed that tumor cellnecrosis caused by CV706 in combination with radiation was significantlygreater than by CV706 (p<0.03) and irradiation perse (p<0.0001). Thisobservation is in agreement with the number of apoptotic cells observedin the treated tumors. The number of apoptotic cells, detected usingTUNEL assay (Milross et al., 2000) in the tumors treated with CV706 andirradiation is 16-fold higher than vehicle, 8.8-fold higher thanirradiation and 3.2-fold higher than CV706.

Secondly, a significant reduction in blood vessel numbers was observedin the tumors treated with CV706 in combination with irradiation.Average number of blood vessel observed at a magnification of 400× intumors treated with vehicle, CV706, radiation or the combination ofCV706 and radiation were 87.5±6.3, 27.5±8.9, 58.5±3.1 and 4.5±1.9,respectively. Significantly reduced numbers of blood vessels in thetumors treated with combination in comparison to CV706 alone orirradiation alone (p<0.01) suggest that the reduction of tumorvascularization may contribute to enhanced tumor regression. It isunclear at this time as to the precise mechanism by which this reductionin blood vessel number is achieved. The possibility for such aneventuality through direct damage of endothelial cells or indirectlythrough the destruction of tumor vasculature by extensive necrosis seemshighly possible. CD31 is expressed constitutively on the surface ofadult and embryonic endothelial cells and has been used as a marker todetect angiogenesis (Giatromanolaki et al., 1997, Clin. Can. Res. 3(12pt 1): 2485-92). Immunohistochemical staining was performed toexamine the effect of treatment on tumor angigenesis by using monoclonalantibody against CD31 (Horak et al., 1992). Tumors treated with CV706followed by irradiation showed a significantly lower level of CD31positive vessel when compared to radiation (p=0.003) or CV706 alone(p=0.03). When compared to untreated mice, CV706/radiation treated miceexhibited significantly lower (4-fold) CD31 positive blood vessel counts(p<0.0001), whereas, radiation treated or CV706 treated mice displayed1.6-fold (p=0.03) or 2.1-fold (p=0.004) lower CD31 positive blood vesselcounts. These observations suggest that CV706 in combination withradiation may be inhibiting tumor angiogenesis to a significant extent.

Finally, treatment employing the combination seems to have a beneficialeffect on the general health of the treated animals in comparison to theindividual treatment. The quality of life of the treated animals seemsto be greatly improved as evidenced by the general appearance andsignificant gain in the body weight. Indeed, animals treated with bothCV706 and irradiation gain 38% more weight than untreated controlanimals, 22% more than CV706 treated animals and 25% more weight thanirradiation treated animals. The combination treatment seems to protectthe animals from the transient weight loss observed in the case ofanimals treated with irradiation alone. TABLE 12 IRES Sequences SEQ IDNO:   A 519 base pair IRES obtainable from encephelomycarditis virus(EMCV). 1 GAC GTCGAC TAATTCCGGTTATTTTCCACCATATTGCCGTCTTTTGGCAA     Sa1I51 TGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGG 101GTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAG 151GAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGAC 201CCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCC 251AAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGC 301CACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAG 351CGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGG 401GATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGG 451TTAAAAAACGTCTAGGCCCCCCGAACCACGGTTACGTGGTTTTCCTTTGA            Sa1I 501AAAACACGATGTCGACGTC SEQ ID NO:   An IRES obtainable from vascularendothelial growth factor (VEGF). 1ACGTAGTCGACAGCGCAGAGGCTTGGGGCAGCCGAGCGGCAGCCAGGCCC        Sail 51CGGCCCGGGCCTCGGTTCCAGAAGGGAGAGGAGCCCGCCAAGGCGCGCAA 101GAGAGCGGGCTGCCTCGCAGTCCGAGCCGGAGAGGGAGCGCGAGCCGCGC 151CGGCCCCGGACGGCCTCCGAAACCATGGTCGACACGTA                                  Sa1I SEQ ID NO:   A 5′UTR region ofHCV. 1 GCCAGCCCCCTGATGGGGGCGACACTCCGCCATGAATCACTCCCCTGTGAGGAACTACTG 61TCTTCACGCAGAAAGCGTCTAGCCATGGCGTTAGTATGAGTGTCGTGCAGCCTCCAGGAC 121CCCCCCTCCCGGGAGAGCCATAGTGGTCTGCGGAACCGGTGAGTACACCGGAATTGCCAG 181GACGACCGGGTCCTTTCTTGGATTAACCCGCTCAATGCCTGGAGATTTGGGCGTGCCCCC 241GCAAGACTGCTAGCCGAGTAGTGTTGGGTCGCGAAAGGCCTTGTGGTACTGCCTGATAGG 301GTGCTTGCGAGTGCCCCGGGAGGTCTCGTAGACCGTGCACC (341) SEQ ID NO:   A 5′UTRregion of BiP SEQ ID NO:4 1CCCGGGGTCACTCCTGCTGGACCTACTCCGACCCCCTAGGCCGGGAGTGAAGGCGGGACT 61TGTGCGGTTACCAGCGGAAATGCCTCGGGGTCAGAAGTCGCAGGAGAGATAGACAGCTGC 121TGAACCAATGGGACCAGCGGATGGGGCGGATGTTATCTACCATTGGTGAACGTTAGAAAC 181GAATAGCAGCCAATGAATCAGCTGGGGGGGCGGAGCAGTGACGTTTATTGCGGAGGGGGC 241CGCTTCGAATCGGCGGCGGCCAGCTTGGTGGCCTGGGCCAATGAACGGCCTCCAACGAGC 301AGGGCCTTCACCAATCGGCGGCCTCCACGACGGGGCTGGGGGAGGGTATATAAGCCGAGT 361AGGCGACGGTGAGGTCGACGCCGGCCAAGACAGCACAGACAGATTGACCTATTGGGGTGT 421TTCGCGAGTGTGAGAGGGAAGCGCCGCGGCCTGTATTTCTAGACCTGCCCTTCGCCTGGT 481TCGTGGCGCCTTGTGACCCCGGGCCCCTGCCGCCTGCAAGTCGAAATTGCGCTGTGCTCC 541TGTGCTACGGCCTGTGGCTGGACTGCCTGCTGCTGCCCAACTGGCTGGCAAGATG (595) SEQ IDNO:   A 5′UTR of PDGF SEQ ID NO:5. 1GTTTGCACCTCTCCCTGCCCGGGTGCTCGAGCTGCCGTTGCAAAGCCAACTTTGGAAAAA 61GTTTTTTGGGGGAGACTTGGGCCTTGAGGTGCCCAGCTCCGCGCTTTCCGATTTTGGGGG 121CTTTCCAGAAAATGTTGCAAAAAAGCTAAGCCGGCGCGCAGAGGAAAACGCCTGTAGCCG 181GCGAGTGAAGACGAACCATCGACTGCCGTGTTCCTTTTCCTCTTGGAGGTTGGAGTCCCC 241TGGGCGCCCCCACACCCCTAGACGCCTCGGCTGGTTCGCGACGCAGCCCCCCGGCCGTGG 301ATGCTGCACTCGGGCTCGGGATCCGCCCAGGTAGCCGGCCTCGGACCCAGGTCCTGCGCC 361CAGGTCCTCCCCTGCCCCCCAGCGACGGAGCCGGGGCCGGGGGCGGCGGCGCCGGGGGCA 421TGCGGGTGAGCCGCGGCTGCAGAGGCCTGAGCGCCTGATCGCCGCGGACCTGAGCCGAGC 481CCACCCCCCTCCCCAGCCCCCCACCCTGGCCGCGGGGGCGGCGCGCTCGATCTACGCGTC 541CGGGGCCCCGCGGGGCCGGGCCCGGAGTCGGCATG (575)

TABLE 13 Literature References For IRES IRES Host Example ReferencePicornavirus HAV Glass et al., 1993. Virol 193: 842-852 EMCV Jang &Wimmer, 1990. Gene Dev 4: 1560-1572 Poliovirus Borman et al., 1994. EMBOJ 13: 3149-3157 HCV and HCV Tsukiyama-Kohara et al., 1992. J Virol 66:1476-1483 pestivirus BVDV Frolov I et al., 1998. RNA. 4: 1418-1435Leishmania LRV-1 Maga et al., 1995. Mol Cell Biol 15: 4884-4889 virusRetroviruses MoMLV Torrent et al., 1996. Hum Gene Ther 7: 603-612 VL30(Harvey murine sarcoma virus) REV Lopez-Lastra et al., 1997. Hum GeneTher 8: 1855-1865 Eukaryotic BiP Macejak & Sarnow, 1991. Nature 353:90-94 mRNA antennapedia Oh et al., 1992. Gene & Dev 6: 1643-1653 mRNAFGF-2 Vagner et al., 1995. Mol Cell Biol 15: 35-44 PDGF-B Bernstein etal., 1997. J Biol Chem 272: 9356-9362 IGFII Teerink et al., 1995.Biochim Biophys Acta 1264: 403-408 eIF4G Gan & Rhoads, 1996. J Biol Chem271: 623-626 VEGF Stein et al., 1998. Mol Cell Biol 18: 3112-3119; Huezet al., 1998. Mol Cell Biol 18: 6178-6190

TABLE 14 TRE Sequences Nucleotide sequence of a human uroplakin II 5′flanking region. Position +1 (the translational start site) is denotedwith an asterisk. SEQ ID NO:_ (number 1 of SEQ ID NO:_ corresponds toposition −2239 with respect to the translational start site). 1TCGATAGGTA CCCACTATAG GGCACGCGTG GTCGACGGCC CGGGCTGGTC 50 51 TGGCAACTTCAAGTGTGGGC CTTTCAGACC GGCATCATCA GTGTTACGGG 100 101 GAAGTCACTAGGAATGCAGA ATTGATTGAG CACGGTGGCT CACACCTGTA 150 151 ATCCCAACACTCTGGGAGGC CAAGGCAGGT GGATCACTTG TGGTCAGGAG 200 201 TTTGAGACCAGCCTGGCCAA CATGGTGAAA CCTCATCTCT ACTAAAAATA 250 251 CAAAAATTAGCTGGGAATGG TGGCACATGC CTATAATCCC AGTTACTCAG 300 301 GAGGCTGAGGCAGGAGAATC ATTTGAACCT GGGAGGCAGA GGTTGCAGTG 350 351 AGCCGAGATCACGCCACTGC ACTCCAGCCT GGGTGACACA GCGAGACTCT 400 401 GTCTCAAAAAAAAAAAAATG CAGAATTTCA GGCTTCACCC CAGACCCACT 450 451 GCATGACTGCATGAGAAGCT GCATCTTAAC AAGATCCCTG GTAATTCATA 500 501 CGCATATTAAATTTGGAGAT GCACTGGCGT AAGACCCTCC TACTCTCTGC 550 551 TTAGGCCCATGAGTTCTTCC TTTACTGTCA TTCTCCACTC ACCCCAAACT 600 601 TTGAGCCTACCCTTCCCACC TTGGCGGTAA GGACACAACC TCCCTCACAT 650 651 TCCTACCAGGACCCTAAGCT TCCCTGGGAC TGAGGAAGAT AGAATAGTTC 700 701 GTGGAGCAAACAGATATACA GCAACAGTCT CTGTACAGCT CTCAGGCTTC 750 751 TGGAAGTTCTACAGCCTCTC CCGACAAAGT ATTCCACTTT CCACAAGTAA 800 801 CTCTATGTGTCTGAGTCTCA GTTTCCACTT TTCTCTCTCT CTCTCTCTCT 850 851 CAACTTTCTGAGACAGAGTT TCACTTAGTC GCCCAGGCTG GAGTGCAGGG 900 901 GCACAATCTCGGCTCACTGC AACCTCCACC TCCTGGGTTC AAGTGTTTCT 950 951 CCTGTCTCAGCCTCCCGAGT AGCTGGGATT ACAGGCACAC ACCACCGCGT 1000 1001 TAGTTTTTGTATTTTTGGTA GAGATGGTGT TTCGCCATAT TGGCCAGGCT 1050 1051 GATCTCGAACTCCTGACCTC AGGTGATCCG CCCACCTCGG CCTCCCAAAG 1100 1101 TGCTGGGATTACAGGCATGA GCCACCACGC CCGGCTGATC TCTTTTCTAT 1150 1151 TTTAATAGAGATCAAACTCT CTGTGTTGCC TAGGCTGGTC TTGAACTCCT 1200 1201 GGCCTCGAGTGATCCTCCCA CCTTGGCCTC CCAAAGTGTT GAGATTACAG 1250 1251 GCATGAGCCACTGTGCCTGG CCTCAGTTCT ACTACAAAAG GAAGCCAGTA 1300 1301 CCAGCTACCACCCAGGGTGG CTGTAGGGCT ACAATGGAGC ACACAGAACC 1350 1351 CCTACCCAGGGCCCGGAAGA AGCCCCGACT CCTCTCCCCT CCCTCTGCCC 1400 1401 AGAACTCCTCCGCTTCTTTC TGATGTAGCC CAGGGCCGGA GGAGGCAGTC 1450 1451 AGGGAAGTTCTGTCTCTTTT TCATGTTATC TTACGAGGTC TCTTTTCTCC 1500 1501 ATTCTCAGTCCAACAAATGG TTGCTGCCCA AGGCTGACTG TGCCCACCCC 1550 1551 CAACCCCTGCTGGCCAGGGT CAATGTCTGT CTCTCTGGTC TCTCCAGAAG 1600 1601 TCTTCCATGGCCACCTTCGT CCCCACCCTC CAGAGGAATC TGAAACCGCA 1650 1651 TGTGCTCCCTGGCCCCCACA GCCCCTGCCT CTCCCAGAGC AGCAGTACCT 1700 1701 AAGCCTCAGTGCACTCCAAG AATTGAAACC CTCAGTCTGC TGCCCCTCCC 1750 1751 CACCAGAATGTTTCTCTCCC ATTCTTACCC ACTCAAGGCC CTTTCAGTAG 1800 1801 CCCCTTGGAGTATTCTCTTC CTACATATCA GGGCAACTTC CAAACTCATC 1850 1851 ACCCTTCTGAGGGGTGGGGG AAAGACCCCC ACCACATCGG GGGAGCAGTC 1900 1901 CTCCAAGGACTGGCCAGTCT CCAGATGCCC GTGCACACAG GAACACTGCC 1950 1951 TTATGCACGGGAGTCCCAGA AGAAGGGGTG ATTTCTTTCC CCACCTTAGT 2000 2001 TACACCATCAAGACCCAGCC AGGGCATCCC CCCTCCTGGC CTGAGGGCCA 2050 2051 GCTCCCCATCCTGAAAAACC TGTCTGCTCT CCCCACCCCT TTGAGGCTAT 2100 2101 AGGGCCCAAGGGGCAGGTTG GACTGGATTC CCCTCCAGCC CCTCCCGCCC 2150 2151 CCAGGACAAAATCAGCCACC CCAGGGGCAG GGCCTCACTT GCCTCAGGAA 2200 2201 CCCCAGCCTGCCAGCACCTA TTCCACCTCC CAGCCCAGCA 2239 Nucleotide sequence of a mouseuroplakin II 5′ flanking region. The translational start site is denotedwith an asterisk. SEQ ID NO:_ (number 1 of SEQ ID NO:6_ corresponds toposition −3592 with respect to the translational start site). 1CTCGAGGATCTCGGCCCTCTTTCTGCATCCTTGTCCTAAATCATTTTCAT 50 51ATCTTGCTAGACCTCAGTTTGAGAGAAACGAACCTTCTCATTTTCAAGTT 100 101GAAAAAAAAAAGAGGTTCAAAGTGGCTCACTCAAAGTTACAAGCCAACAC 150 151TCACCACTACGAGTACAATGGCCACCATTAGTGCTGGCATGCCCCAGGAG 200 201ACAGGCATGCATATTATTCTAGATGACTGGGAGGCAGAGGGGTGGCCTAG 250 251TGAGGTCAGACTGTGGACAGATCAGGCAGATGTGGGTTCTGATCCCAATT 300 301CCTCAGGCCGCAGAACTACTGTGGTTCAAGAAGGGGACAAAACGACTGCA 350 351GTCCGGAACAGGAGGTCCATTTGAGAGCTGACTGAGCAGAAGAGGAAAGT 400 401GAAGAACTTCTGGGGCAAGAGCTTACCCTACTTTACAGCTTTGTTGTCTT 450 451CTTTACTCCAGGGGCGTCCCTGGTACTCAGTAAATGTCTGTTGGCTTGAG 500 501GAACATATGTGTAAGGAGGAAGGAGAGGGAACTTGAGGGAGTTAAGACTC 550 551AAGAATCAATCAAGGAGAGGACAGCAGAGAAGACAGGGTTTGGGAGAGAG 600 601ACTCCAGACATTGGCCCTGGTTCCCTTCTTGGCCACTGTGAAACCCTCCA 650 651GAGGAACTGAGTGCTGTGGCTTTAAATGATCTCAGCACTGTCAGTGAAGC 700 701GCTCTGCTCAAAGAGTTATCCTCTTGCTCCTGTGCCGGGGCCTCCCCCTC 750 751CTCTCAGCTCCCAAACCCTTCTCAGCCACTGTGATGGCATAATTAGATGC 800 801GAGAGCTCAGACCGTCAGGTCTGCTCCAGGAACCACCCATTTTCCCCAAC 850 851CCCAGAGAAAGGTCCTAGTGGAAAAGTGGGGGCCACTGAAGGGCTGATGG 900 900GGTTCTGTCCTTTCCCCCATGCTGGGTGGACTTAAAGTCTGCGATGTGTG 950 951TAGGGGGTAGAAGACAACAGAACCTGGGGGCTCCGGCTGGGAGCAGGAGG 1000 1001AACTCTCACCAGACGATCTCCAAATTTACTGTGCAATGGACGATCAGGAA 1050 1051ACTCGTTCAGATGTAGCTTCTGATACAGTGGGTCTGAGGTAAAACCCGAA 1100 1101ACTTAATTTCTTTCAAAAATTTAAAGTTGCATTTATTATTTTATATGTGT 1150 1151GCCCATATGTGTGCCACAGTGTCTATGTGGAGGTCAGAGGGCAAGTTGTG 1200 1201GGCATTGGCTCTCTCCTTTCATAATGTGGCTTCTGGGGACCAAAATGTCA 1250 1251GGCATGGTGGCAAGAGCTTTTACCTGTTGAGCCATCTCATGGTTTCGTAA 1300 1301AACTTCCTATGACGCTTACAGGTAACGCAGAGACACAGACTCACATTTGG 1350 1351AGTTAGCAGATGCTGTATTGGTGTAAACACTCATACACAGACACACACAC 1400 1401ATACTCATACACACACACACACACTTATCACATGCACACACATACTCGTA 1450 1451TACACACAGACACACACACATGCACTCTCACATTCACATATTCATACACA 1500 1501TCCACACACACACTCATCCACACACACAGACACACATACTCATCCACACA 1550 1551CACACACACACATACTCATACACACACACAGACACACATACTCATACACA 1600 1601CACACAGACACACACATATAATCATACATACACAGACACACTCATACATG 1650 1651TGCACACACACACTCATCCACACACACACACTCATACACACACACACTCA 1700 1701TACACACACACACTCATACACACACACACGAGGTTTTTCTCAGGCTGCCT 1750 1751TTGGGTGGAGACTGGAACTGATTTCTGTTTTTCAGCTCCTTGGCTTTTTG 1800 1801TCCCTTTAGATGAGATCTCCTCCTCACTTTACACACAGAAAGATCACACA 1850 1851CGAGGGAGAACTGGCGGTGCGGAAGAGGGCTACACGGTAGGGTGTCAGGG 1900 1901TCAGGAGATCTTCCTGGCAAGTCTCAAACCTCCACATAGCACAGTGTTTA 1950 1951CGTGAGGATTTAGGAGGAATCAGGAAGAGGATTGGTTTACTGCAGAGCAG 2000 2001ACCATATAGGTCCACTCCTAAGCCCCATTTGAAATTAGAAGTGAGACAGT 2050 2051GTGGGATAAAAAGAGCAGATCTCTGGTCACATTTTTAAAGGGATATGAGG 3000 2101GTCCTGTGCCTTTAAGCCTTCCCATCTCCCTCCAATCCCCCCTCACCTTC 2150 2151CCCACCCTAACCCTCCCCAGGTTTCTGGAGGAGCAGAGTTGCGTCTTCTC 2200 2201CCTGCCCTGCCGAGCTGCTCACTGGCTGCTCTAGAGGCTGTGCTTTGCGG 2250 2251TCTCCATGGAAACCATTAGTTGCTAAGCAACTGGAGCATCATCTGTGCTG 2300 2301AGCTCAGGTCCTATCGAGTTCACCTAGCTGAGACACCCACGCCCCTGCAG 2350 2351CCACTTTGCAGTGACAAGCCTGAGTCTCAGGTTCTGCATCTATAAAAACG 2400 2401AGTAGCCTTTCAGGAGGGCATGCAGAGCCCCCTGGCCAGCGTCTAGAGGA 2450 2451GAGGTGACTGAGTGGGGCCATGTCACTCGTCCATGGCTGGAGAACCTCCA 2500 2501TCAGTCTCCCAGTTAGCCTGGGGCAGGAGAGAACCAGAGGAGCTGTGGCT 2550 2551GCTGATTGGATGATTTACGTACCCAATCTGTTGTCCCAGGCATCGAACCC 2600 2601CAGAGCGACCTGCACACATGCCACCGCTGCCCCGCCCTCCACCTCCTCTG 2650 2651CTCCTGGTTACAGGATTGTTTTGTCTTGAAGGGTTTTGTTGTTGCTACTT 2700 2701TTTGCTTTGTTTTTTCTTTTTTAACATAAGGTTTCTCTGTGTAGCCCTAG 2750 2751CTGTCCTGGAACTCACTCTGTAGACCAGGCTGGCCTCAAACTCAGAAATC 2800 2801CACCTTCCTCCCAAGTGCTGGGATTAAAGGCATTCGCACCATCGCCCAGC 2850 2851CCCCGGTCTTGTTTCCTAAGGTTTTCCTGCTTTACTCGCTACCCGTTGCA 2900 2901CAACCGCTTGCTGTCCAAGTCTGTTTGTATCTACTCCACCGCCCACTAGC 2950 2951CTTGCTGGACTGGACCTACGTTTACCTGGAAGCCTTCACTAACTTCCCTT 3000 3001GTCTCCACCTTCTGGAGAAATCTGAAGGCTCACACTGATACCCTCCGCTT 3050 3051CTCCCAGAGTCGCAGTTTCTTAGGCCTCAGTTAAATACCAGAATTGGATC 3100 3101TCAGGCTCTGCTATCCCCACCCTACCTAACCAACCCCCTCCTCTCCCATC 3150 3151CTTACTAGCCAAAGCCCTTTCAACCCTTGGGGCTTTTCCTACACCTACAC 3200 3201ACCAGGGCAATTTTAGAACTCATGGCTCTCCTAGAAAACGCCTACCTCCT 3250 3251TGGAGACTGACCCTCTACAGTCCAGGAGGCAGACACTCAGACAGAGGAAC 3300 3301TCTGTCCTTCAGTCGCGGGAGTTCCAGAAAGAGCCATACTCCCCTGCAGA 3350 3351GCTAACTAAGCTGCCAGGACCCAGCCAGAGCATCCCCCTTTAGCCGAGGG 3400 3401CCAGCTCCCCAGAATGAAAAACCTGTCTGGGGCCCCTCCCTGAGGCTACA 3450 3451GTCGCCAAGGGGCAAGTTGGACTGGATTCCCAGCAGCCCCTCCCACTCCG 3500 3501AGACAAAATCAGCTACCCTGGGGCAGGCCTCATTGGCCCCAGGAAACCCC 3550 3551AGCCTGTCAGCACCTGTTCCAGGATCCAGTCCCAGCGCAGTA 3592 AFP-TRE SEQ ID NO:_(—) 1GCATTGCTGTGAACTCTGTACTTAGGACTAAACTTTGAGCAATAACACACATAGATTGAG 61GATTGTTTGCTGTTAGCATACAAACTCTGGTTCAAAGCTCCTCTTTATTGCTTGTCTTGG 121AAAATTTGCTGTTCTTCATGGTTTCTCTTTTCACTGCTATCTATTTTTCTCAACCACTCA 181CATGGCTACAATAACTGTCTGCAAGCTTATGATTCCCAAATATCTATCTCTAGCCTCAAT 241CTTGTTCCAGAAGATAAAAAGTAGTATTCAAATGCACATCAACGTCTCCACTTGGAGGGC 301TTAAAGACGTTTCAACATACAAACCGGGGAGTTTTGCCTGGAATGTTTCCTAAAATGTGT 361CCTGTAGCACATAGGGTCCTCTTGTTCCTTAAAATCTAATTACTTTTAGCCCAGTGCTCA 421TCCCACCTATGGGGAGATGAGAGTGAAAAGGGAGCCTGATTAATAATTACACTAAGTCAR 481TAGGCATAGAGCCAGGACTGTTTGGGTAAACTGGTCACTTTATCTTAAAcTAAATATATC 541CAAAACTGAACATGTACTTAGTTACTAAGTCTTTGACTTTATCTCATTCATACCACTCAG 601CTTTATCCAGGCCACTTATGAGCTCTGTGTCCTTGAACATAAAATACAAATAACCGCTAT 661GCTGTTAATTATTGGCAAATGTCCCATTTTCAACCTAAGGAAATACCATAAAGTAACAGA 721TATACCAACAAAAGGTTACTAGTTAACAGGCATTGCCTGAAAAGAGTATAAAAGAATTTC 781AGCATGATTTTCCATATTGTGCTTCCACCACTGCCAATAACA (822) Probasin-TRE SEQ IDNO:_(—)   −426 5′-AAGCTTCCACAAGTGCATTTAGCCTCTCCAGTATTGCTGATGAATCCACAGTTCAGGTTCAATGGCGTTCAAAACTTGATCAAAAATGACCAGACTTTATATTTACACCAACATCTATCTGATTGGAGGAATGGATAATAGTCATCATGTTTAAACATCTACCATTCCAGTTAAGAAAATATGATAGCATCTTGTTCTTAGTCTTTTTCTTA                                    ARE -1ATAGGGACATAAAGCCCACAAATAAAAATATGCCTGAAGAATGGGACAGGCATTGGGCATTGTCCATGCCTAGTAAAGTACTCCAAGAACCTATTTGTATACTA                              ARE-2GATGACACAATGTCAATGTCTGTGTACAACTGCCAACTGGGATGCAAGACAC TGCCCATG CCAATCATCCTGAAAAGCAGC TATAAAAA GCAGGAAGCTACTCT        CAATbox               TATAA box     +1                        +28GCACCTTGTCAGTAGGTCCAGATACCTACAG-3′ Transcription site Tyrosinase-TRE SEQID NO:_(—) PinA1 end 1CCGGTTGAAAATGATAAGTTGAATTCTGTCTTCGAGAACATAGAAAAGAA 51TTATGAAATGCCAACATGTGGTTACAAGTAATGCAGACCCAAGGCTCCCC 101AGGGACAAGAAGTCTTGTGTTAACTCTTTGTGGCTCTGAAAGAAAGAGAG 151AGAGAAAAGATTAAGCCTCCTTGTGGAGATCATGTGATGACTTCCTGATT 201CCAGCCAGAGCGAGCATTTCCATGGAAACTTCTCTTCCTCTTCACTCGAG 251ATTACTAACCTTATTGTTAATATTCTAACCATAAGAATTAAACTATTAAT 301GGTGAATAGAGTTTTTCACTTTAACATAGGCCTATCCCACTGGTGGGATA 351CGAGCCAATTCGAAAGAAAAAGTCAGTCATGTGCTTTTCAGAGGATGAAA 401GCTTAAGATAAAGACTAAAAGTGTTTGATGCTGGAGGTGGGAGTGGTATT 451ATATAGGTCTCAGCCAAGACATGTGATAATCACTGTAGTAGTAGCTGGAA 501AGAGAAATCTGTGACTCCAATTAGCCAGTTCCTGCAGACCTTGTGA PinA1 end Human glandularkallikrein-TRE SEQ ID NO:_(—) gaattcagaa ataggggaag gttgaggaaggacactgaac tcaaagggga tacagtgatt 60 ggtttatttg tcttctcttc acaacattggtgctggagga attcccaccc tgaggttatg 120 aagatgtctg aacacccaac acatagcactggagatatga gctcgacaag agtttctcag 180 ccacagagat tcacagccta gggcaggaggacactgtacg ccaggcagaa tgacatggga 240 attgcgctca cgattggctt gaagaagcaaggactgtggg aggtgggctt tgtagtaaca 300 agagggcagg gtgaactctg attcccatgggggaatgtga tggtcctgtt acaaattttt 360 caagctggca gggaataaaa cccattacggtgaggacctg tggagggcgg ctgccccaac 420 tgataaagga aatagccagg tgggggcctttcccattgta ggggggacat atctggcaat 480 agaagccttt gagacccttt agggtacaagtactgaggca gcaaataaaa tgaaatctta 540 tttttcaact ttatactgca tgggtgtgaagatatatttg tttctgtaca gggggtgagg 600 gaaaggaggg gaggaggaaa gttcctgcaggtctggtttg gtcttgtgat ccagggggtc 660 ttggaactat ttaaattaaa ttaaattaaaacaagcgact gttttaaatt aaattaaatt 720 aaattaaatt ttactttatt ttatcttaagttctgggcta catgtgcagg acgtgcagct 780 ttgttacata ggtaaacgtg tgccatggtggtttgctgta cctatcaacc catcacctag 840 gtattaagcc cagcatgcat tagctgtttttcctgacgct ctccctctcc ctgactccca 900 caacaggccc cagtgtgtgt tgttcccctccctgtgtcca tgtgttctca ttgttcagct 960 cccacttata agtgagaaca tgtggtgtttggttttctgt ttctgtgtta gtttgctgag 1020 gataatggct tccacctcca tccatgttcctgcaaaggac gtgatcttat tcttttttat 1080 ggttgcatag aaattgtttt tacaaatccaattgatattg tatttaatta caagttaatc 1140 taattagcat actagaagag attacagaagatattaggta cattgaatga ggaaatatat 1200 aaaataggac gaaggtgaaa tattaggtaggaaaagtata atagttgaaa gaagtaaaaa 1260 aaaatatgca tgagtagcag aatgtaaaagaggtgaagaa cgtaatagtg actttttaga 1320 ccagattgaa ggacagagac agaaaaattttaaggaattg ctaaaccatg tgagtgttag 1380 aagtacagtc aataacatta aagcctcaggaggagaaaag aataggaaag gaggaaatat 1440 gtgaataaat agtagagaca tgtttgatggattttaaaat atttgaaaga cctcacatca 1500 aaggattcat accgtgccat tgaagaggaagatggaaaag ccaagaagcc agatgaaagt 1560 tagaaatatt attggcaaag cttaaatgttaaaagtccta gagagaaagg atggcagaaa 1620 tattggcggg aaagaatgca gaacctagaatataaattca tcccaacagt ttggtagtgt 1680 gcagctgtag ccttttctag ataatacactattgtcatac atcgcttaag cgagtgtaaa 1740 atggtctcct cactttattt atttatatatttatttagtt ttgagatgga gcctcgctct 1800 gtctcctagg ctggagtgca atagtgcgataccactcact gcaacctctg cctcctctgt 1860 tcaagtgatt ttcttacctc agcctcccgagtagctggga ttacaggtgc gtgccaccac 1920 acccggctaa tttttgtatt ttttgtagagacggggtttt gccatgttgg ccaggctggt 1980 cttgaactcc tgacatcagg tgatccacctgccttggcct cctaaagtgc tgggattaca 2040 ggcatgagcc accgtgccca accactttatttatttttta tttttatttt taaatttcag 2100 cttctatttg aaatacaggg ggcacatatataggattgtt acatgggtat attgaactca 2160 ggtagtgatc atactaccca acaggtaggttttcaaccca ctccccctct tttcctcccc 2220 attctagtag tgtgcagtgt ctattgttctcatgtttatg tctatgtgtg ctccaggttt 2280 agctcccacc tgtaagtgag aacgtgtggtatttgatttt ctgtccctgt gttaattcac 2340 ttaggattat ggcttccagc tccattcatattgctgtaaa ggatatgatt catttttcat 2400 ggccatgcag tattccatat tgcgtatagatcacattttc tttctttttt ttttttgaga 2460 cggagtcttg ctttgctgcc taggctggagtgcagtagca cgatctcggc tcactgcaag 2520 cttcacctcc ggggttcacg tcattcttctgtctcagctt cccaagtagc tgggactaca 2580 ggcgcccgcc accacgtccg gctaatttttttgtgtgttt ttagtagaga tgggggtttc 2640 actgtgttag ccaggatggt cttgatctcctgaccttgtg gtccacctgc ctcggtctcc 2700 caaagtgctg ggattacagg ggtgagccactgcgcccggc ccatatatac cacattttct 2760 ttaaccaatc caccattgat gggcaactaggtagattcca tggattccac agttttgcta 2820 ttgtgtgcag tgtggcagta gacatatgaatgaatgtgtc tttttggtat aatgatttgc 2880 attcctttgg gtatacagtc attaataggagtgctgggtt gaacggtggc tctgtttaaa 2940 attctttgag aattttccaa actgtttgccatagagagca aactaattta catttccacg 3000 aacagtatat aagcattccc ttttctccacagctttgtca tcatggtttt tttttttctt 3060 tattttaaaa aagaatatgt tgttgttttcccagggtaca tgtgcaggat gtgcaggttt 3120 gttacatagg tagtaaacgt gagccatggtggtttgctgc acctgtcaac ccattacctg 3180 ggtatgaagc cctgcctgca ttagctcttttccctaatgc tctcactact gccccaccct 3240 caccctgaca gggcaaacag acaacctacagaatgggagg aaatttttgc aatctattca 3300 tctgacaaag gtcaagaata tccagaatctacaaggaact taagcaaatt tttacttttt 3360 aataatagcc actctgactg gcgtgaaatggtatctcatt gtggttttca tttgaatttc 3420 tctgatgatc agtgacgatg agcattttttcatatttgtt ggctgcttgt acgtcttttg 3480 agaagtgtct cttcatgcct tttggccactttaatgggat tattttttgc tttttagttt 3540 aagttcctta tagattctgg atattagacttcttattgga tgcatagttt gtgaatactc 3600 tcttccattc tgtaggttgt ctgtttactctattgatggc ttcttttgct gtgccgaagc 3660 atcttagttt aattagaaac cacctgccaatttttgtttt tgttgcaatt gcttttgggg 3720 acttagtcat aaactctttg ccaaggtctgggtcaagaag agtatttcct aggttttctt 3780 ctagaatttt gaaagtctga atgtaaacatttgcattttt aatgcatctt gagttagttt 3840 ttgtatatgt gaaaggtcta ctctcattttctttccctct ttctttcttt ctttcttttc 3900 tttctttctt tctttctttc tttctttctttctttctttc tttctttttg tccttctttc 3960 tttctttctt tctctttctt tctctctttctttttttttt ttgatggagt attgctctgt 4020 tgcccaggct gcagtgcagc ggcacgatctcggctcactg caacctctgc ctcctgggtt 4080 caactgattc tcctgcatca gccttccaagtagctgggat tataggcgcc cgccaccacg 4140 cccgactaat ttttgtattt ttagtagagacggggttgtg ccatgttggc caggctggtt 4200 tgaaactcct gacctcaaac gatctgcctgccttggcctc ccaaagtgct gggattacag 4260 gtgtgagcca ctgtgcccag ccaagaatgtcattttctaa gaggtccaag aacctcaaga 4320 tattttggga ccttgagaag agaggaattcatacaggtat tacaagcaca gcctaatggc 4380 aaatctttgg catggcttgg cttcaagactttaggctctt aaaagtcgaa tccaaaaatt 4440 tttataaaag ctccagctaa gctaccttaaaaggggcctg tatggctgat cactcttctt 4500 gctatacttt acacaaataa acaggccaaatataatgagg ccaaaattta ttttgcaaat 4560 aaattggtcc tgctatgatt tactcttggtaagaacaggg aaaatagaga aaaatttaga 4620 ttgcatctga cctttttttc tgaatttttatatgtgccta caatttgagc taaatcctga 4680 attattttct ggttgcaaaa actctctaaagaagaacttg gttttcattg tcttcgtgac 4740 acatttatct ggctctttac tagaacagctttcttgtttt tggtgttcta gcttgtgtgc 4800 cttacagttc tactcttcaa attattgttatgtgtatctc atagttttcc ttcttttgag 4860 aaaactgaag ccatggtatt ctgaggactagagatgactc aacagagctg gtgaatctcc 4920 tcatatgcaa tccactgggc tcgatctgcttcaaattgct gatgcactgc tgctaaagct 4980 atacatttaa aaccctcact aaaggatcagggaccatcat ggaagaggag gaaacatgaa 5040 attgtaagag ccagattcgg ggggtagagtgtggaggtca gagcaactcc accttgaata 5100 agaaggtaaa gcaacctatc ctgaaagctaacctgccatg gtggcttctg attaacctct 5160 gttctaggaa gactgacagt ttgggtctgtgtcattgccc aaatctcatg ttaaattgta 5220 atccccagtg ttcggaggtg ggacttggtggtaggtgatt cggtcatggg agtagatttt 5280 cttctttgtg gtgttacagt gatagtgagtgagttctcgt gagatctggt catttaaaag 5340 tgtgtggccc ctcccctccc tctcttggtcctcctactgc catgtaagat acctgctcct 5400 gctttgcctt ctaccataag taaaagccccctgaggcctc cccagaagca gatgccacca 5460 tgcttcctgt acagcctgca gaaccatcagccaattaaac ctcttttctg tataaattac 5520 cagtcttgag tatctcttta cagcagtgtgagaacggact aatacaaggg tctccaaaat 5580 tccaagttta tgtattcttt cttgccaaatagcaggtatt taccataaat cctgtcctta 5640 ggtcaaacaa ccttgatggc atcgtacttcaattgtctta cacattcctt ctgaatgact 5700 cctcccctat ggcatataag ccctgggtcttgggggataa tggcagaggg gtccaccatc 5760 ttgtctggct gccacctgag acacggacatggcttctgtt ggtaagtctc tattaaatgt 5820 ttctttctaa gaaactggat ttgtcagcttgtttctttgg cctctcagct tcctcagact 5880 ttggggtagg ttgcacaacc ctgcccaccacgaaacaaat gtttaatatg ataaatatgg 5940 atagatataa tccacataaa taaaagctcttggagggccc tcaataattg ttaagagtgt 6000 aaatgtgtcc aaagatggaa aatgtttgagaactactgtc ccagagattt tcctgagttc 6060 tagagtgtgg gaatatagaa cctggagcttggcttcttca gcctagaatc aggagtatgg 6120 ggctgaagtc tgaagcttgg cttcagcagtttggggttgg cttccggagc acatatttga 6180 catgttgcga ctgtgatttg gggtttggtatttgctctga atcctaatgt ctgtccttga 6240 ggcatctaga atctgaaatc tgtggtcagaattctattat cttgagtagg acatctccag 6300 tcctggttct gccttctagg gctggagtctgtagtcagtg acccggtctg gcatttcaac 6360 ttcatataca gtgggctatc ttttggtccatgtttcaacc aaacaaccga ataaaccatt 6420 agaacctttc cccacttccc tagctgcaatgttaaaccta ggatttctgt ttaataggtt 6480 catatgaata atttcagcct gatccaactttacattcctt ctaccgttat tctacaccca 6540 ccttaaaaat gcattcccaa tatattccctggattctacc tatatatggt aatcctggct 6600 ttgccagttt ctagtgcatt aacatacctgatttacattc ttttacttta aagtggaaat 6660 aagagtccct ctgcagagtt caggagttctcaagatggcc cttacttctg acatcaattg 6720 agatttcaag ggagtcgcca agatcatcctcaggttcagt gattgctggt agccctcata 6780 taactcaatg aaagctgtta tgctcatggctatggtttat tacagcaaaa gaatagagat 6840 gaaaatctag caagggaaga gttgcatggggcaaagacaa ggagagctcc aagtgcagag 6900 attcctgttg ttttctccca gtggtgtcatggaaagcagt atcttctcca tacaatgatg 6960 tgtgataata ttcagtgtat tgccaatcagggaactcaac tgagccttga ttatattgga 7020 gcttggttgc acagacatgt cgaccaccttcatggctgaa ctttagtact tagcccctcc 7080 agacgtctac agctgatagg ctgtaacccaacattgtcac cataaatcac attgttagac 7140 tatccagtgt ggcccaagct cccgtgtaaacacaggcact ctaaacaggc aggatatttc 7200 aaaagcttag agatgacctc ccaggagctgaatgcaaaga cctggcctct ttgggcaagg 7260 agaatccttt accgcacact ctccttcacagggttattgt gaggatcaaa tgtggtcatg 7320 tgtgtgagac accagcacat gtctggctgtggagagtgac ttctatgtgt gctaacattg 7380 ctgagtgcta agaaagtatt aggcatggctttcagcactc acagatgctc atctaatcct 7440 cacaacatgg ctacagggtg ggcactactagcctcatttg acagaggaaa ggactgtgga 7500 taagaagggg gtgaccaata ggtcagagtcattctggatg caaggggctc cagaggacca 7560 tgattagaca ttgtctgcag agaaattatggctggatgtc tctgccccgg aaagggggat 7620 gcactttcct tgacccccta tctcagatcttgactttgag gttatctcag acttcctcta 7680 tgataccagg agcccatcat aatctctctgtgtcctctcc ccttcctcag tcttactgcc 7740 cactcttccc agctccatct ccagctggccaggtgtagcc acagtaccta actctttgca 7800 gagaactata aatgtgtatc ctacaggggagaaaaaaaaa aagaactctg aaagagctga 7860 cattttaccg acttgcaaac acataagctaacctgccagt tttgtgctgg tagaactcat 7920 gagactcctg ggtcagaggc aaaagattttattacccaca gctaaggagg cagcatgaac 7980 tttgtgttca catttgttca ctttgccccccaattcatat gggatgatca gagcagttca 8040 ggtggatgga cacaggggtt tgtggcaaaggtgagcaacc taggcttaga aatcctcaat 8100 cttataagaa ggtactagca aacttgtccagtctttgtat ctgacggaga tattatcttt 8160 ataattgggt tgaaagcaga cctactctggaggaacatat tgtatttatt gtcctgaaca 8220 gtaaacaaat ctgctgtaaa atagacgttaactttattat ctaaggcagt aagcaaacct 8280 agatctgaag gogataccat cttgcaaggctatctgctgt acaaatatgc ttgaaaagat 8340 ggtccagaaa agaaaacggt attattgcctttgctcagaa gacacacaga aacataagag 8400 aaccatggaa aattgtctcc caacactgttcacccagagc cttccactct tgtctgcagg 8460 acagtcttaa catcccatca ttagtgtgtctaccacatct ggcttcaccg tgcctaacca 8520 agatttctag gtccagttcc ccaccatgtttggcagtgcc ccactgccaa ccccagaata 8580 agggagtgct cagaattccg aggggacatgggtggggatc agaacttctg ggcttgagtg 8640 cagagggggc ccatactcct tggttccgaaggaggaagag gctggaggtg aatgtccttg 8700 gaggggagga atgtgggttc tgaactcttaaatccccaag ggaggagact ggtaaggtcc 8760 cagcttccga ggtactgacg tgggaatggcctgagaggtc taagaatccc gtatcctcgg 8820 gaaggagggg ctgaaattgt gaggggttgagttgcagggg tttgttagct tgagactcct 8880 tggtgggtcc ctgggaagca aggactggaaccattggctc cagggtttgg tgtgaaggta 8940 atgggatctc ctgattctca aagggtcagaggactgagag ttgcccatgc tttgatcttt 9000 ccatctactc cttactccac ttgagggtaatcacctactc ttctagttcc acaagagtgc 9060 gcctgcgcga gtataatctg cacatgtgccatgtcccgag gcctggggca tcatccactc 9120 atcattcagc atctgcgcta tgcgggcgaggccggcgcca tgacgtcatg tagctgcgac 9180 tatccctgca gcgcgcctct cccgtcacgtcccaaccatg gagctgtgga cgtgcgtccc 9240 ctggtggatg tggcctgcgt ggtgccaggccggggcctgg tgtccgataa agatcctaga 9300 accacaggaa accaggactg aaaggtgctagagaatggcc atatgtcgct gtccatgaaa 9360 tctcaaggac ttctgggtgg agggcacaggagcctgaact tacgggtttg ccccagtcca 9420 ctgtcctccc aagtgagtct cccagatacgaggcactgtg ccagcatcag cttcatctgt 9480 accacatctt gtaacaggga ctacccaggaccctgatgaa caccatggtg tgtgcaggaa 9540 gagggggtga aggcatggac tcctgtgtggtcagagccca gagggggcca tgacgggtgg 9600 ggaggaggct gtggactggc tcgagaagtgggatgtggtt gtgtttgatt tcctttggcc 9660 agataaagtg ctggatatag cattgaaaacggagtatgaa gaccagttag aatggagggt 9720 caggttggag ttgagttaca gatggggtaaaattctgctt cggatgagtt tggggattgg 9780 caatctaaag gtggtttggg atggcatggctttgggatgg aaataggttt gtttttatgt 9840 tggctgggaa gggtgtgggg attgaattggggatgaagta ggtttagttt tggagataga 9900 atacatggag ctggctattg catgcgaggatgtgcattag tttggtttga tctttaaata 9960 aaggaggcta ttagggttgt cttgaattagattaagttgt gttgggttga tgggttgggc 10020 ttgtgggtga tgtggttgga ttgggctgtgttaaattggt ttgggtcagg ttttggttga 10080 ggttatcatg gggatgagga tatgcttgggacatggattc aggtggttct cattcaagct 10140 gaggcaaatt tcctttcaga cggtcattccagggaacgag tggttgtgtg ggggaaatca 10200 ggccactggc tgtgaatatc cctctatcctggtcttgaat tgtgattatc tatgtccatt 10260 ctgtctcctt cactgtactt ggaattgatctggtcattca gctggaaatg ggggaagatt 10320 ttgtcaaatt cttgagacac agctgggtctggatcagcgt aagccttcct tctggtttta 10380 ttgaacagat gaaatcacat tttttttttcaaaatcacag aaatcttata gagttaacag 10440 tggactctta taataagagt taacaccaggactcttattc ttgattcttt tctgagacac 10500 caaaatgaga tttctcaatg ccaccctaattctttttttt tttttttttt tttttgagac 10560 acagtctggg tcttttgctc tgtcactcaggctggagcgc agtggtgtga tcatagctca 10620 ctgaaccctt gacctcctgg acttaagggatcctcctgct tcagcctcct gagtagatgg 10680 ggctacaggt gcttgccacc acacctggctaattaaattt tttttttttt tttgtagaga 10740 aagggtctca ctttgttgcc ctggctgatcttgaacttct gacttcaagt gattcttcag 10800 ccttggactc ccaaagcact gggattgctggcatgagcca ctcaccgtgc ctggcttgca 10860 gcttaatctt ggagtgtata aacctggctcctgatagcta gacatttcag tgagaaggag 10920 gcattggatt ttgcatgagg acaattctgacctaggaggg caggtcaaca ggaatccccg 10980 ctgtacctgt acgttgtaca ggcatggagaatgaggagtg aggaggccgt accggaaccc 11040 catattgttt agtggacatt ggattttgaaataataggga acttggtctg ggagagtcat 11100 atttctggat tggacaatat gtggtatcacaaggttttat gatgagggag aaatgtatgt 11160 ggggaaccat tttctgagtg tggaagtgcaagaatcagag agtagctgaa tgccaacgct 11220 tctatttcag gaacatggta agttggaggtccagctctcg ggctcagacg ggtataggga 11280 ccaggaagtc tcacaatccg atcattctgatatttcaggg catattaggt ttggggtgca 11340 aaggaagtac ttgggactta ggcacatgagactttgtatt gaaaatcaat gattggggct 11400 ggccgtggtg ctcacgcctg taatctcatcactttgggag accgaagtgg gaggatggct 11460 tgatctcaag agttggacac cagcctaggcaacatggcca gaccctctct ctacaaaaaa 11520 attaaaaatt agctggatgt ggtggtgcatgcttgtggtc tcagctatcc tggaggctga 11580 gacaggagaa tcggttgagt ctgggagttcaaggctacag ggagctgcga tcacgccgct 11640 gcactccagc ctgggaaaca gagtgagactgtctcagaat ttttttaaaa aagaatcagt 11700 gatcatccca acccctgttg ctgttcatcctgagcctgcc ttctctggct ttgttcccta 11760 gatcacatct ccatgatcca taggccctgcccaatctgac ctcacaccgt gggaatgcct 11820 ccagactgat ctagtatgtg tggaacagcaagtgctggct ctccctcccc ttccacagct 11880 ctgggtgtgg gagggggttg tccagcctccagcagcatgg ggagggcctt ggtcagcatc 11940 taggtgccaa cagggcaagg gcggggtcctggagaatgaa ggctttatag ggctcctcag 12000 ggaggccccc cagccccaaa ctgcaccacctggccgtgga caccggt 12047 HRE-TRE SEQ ID NO:_(—) ccccgagg cagtgcatgaggctcagg gcgtgcgt gagtcgcagcgagaccccg gggtgcag gccgga PSA-TRE SEQ IDNO:_(—) aagcttctag ttttcttttc ccggtgacat cgtggaaagc actagcatctctaagcaatg 60 atctgtgaca atattcacag tgtaatgcca tccagggaac tcaactgagccttgatgtcc 120 agagattttt gtgttttttt ctgagactga gtctcgctct gtgccaggctggagtgcagt 180 ggtgcaacct tggctcactg caagctccgc ctcctgggtt cacgccattctcctgcctca 240 gcctcctgag tagctgggac tacaggcacc cgccaccacg cctggctaatttttttgtat 300 ttttagtaga gatggggttt cactgtgtta gccaggatgg tctcagtctcctgacctcgt 360 gatctgccca ccttggcctc ccaaagtgct gggatgacag gcgtgagccaccgcgcctgg 420 ccgatatcca gagatttttt ggggggctcc atcacacaga catgttgactgtcttcatgg 480 ttgactttta gtatccagcc cctctagaaa tctagctgat atagtgtggctcaaaacctt 540 cagcacaaat cacaccgtta gactatctgg tgtggcccaa accttcaggtgaacaaaggg 600 actctaatct ggcaggatac tccaaagcat tagagatgac ctcttgcaaagaaaaagaaa 660 tggaaaagaa aaagaaagaa aggaaaaaaa aaaaaaaaaa gagatgacctctcaggctct 720 gaggggaaac gcctgaggtc tttgagcaag gtcagtcctc tgttgcacagtctccctcac 780 agggtcattg tgacgatcaa atgtggtcac gtgtatgagg caccagcacatgcctggctc 840 tggggagtgc cgtgtaagtg tatgcttgca ctgctgaatg gctgggatgtgtcagggatt 900 atcttcagca cttacagatg ctcatctcat cctcacagca tcactatgggatgggtatta 960 ctggcctcat ttgatggaga aagtggctgt ggctcagaaa ggggggaccactagaccagg 1020 gacactctgg atgctgggga ctccagagac catgaccact caccaactgcagagaaatta 1080 attgtggcct gatgtccctg tcctggagag ggtggaggtg gaccttcactaacctcctac 1140 cttgaccctc tcttttaggg ctctttctga cctccaccat ggtactaggaccccattgta 1200 ttctgtaccc tcttgactct atgaccccca ccgcccactg catccagctgggtcccctcc 1260 tatctctatt cccagctggc cagtgcagtc tcagtgccca cctgtttgtcagtaactctg 1320 aaggggctga cattttactg acttgcaaac aaataagcta actttccagagttttgtgaa 1380 tgctggcaga gtccatgaga ctcctgagtc agaggcaaag gcttttactgctcacagctt 1440 agcagacagc atgaggttca tgttcacatt agtacacctt gccccccccaaatcttgtag 1500 ggtgaccaga gcagtctagg tggatgctgt gcagaagggg tttgtgccactggtgagaaa 1560 cctgagatta ggaatcctca atcttatact gggacaactt gcaaacctgctcagcctttg 1620 tctctgatga agatattatc ttcatgatct tggattgaaa acagacctactctggaggaa 1680 catattgtat cgattgtcct tgacagtaaa caaatctgtt gtaagagacattatctttat 1740 tatctaggac agtaagcaag cctggatctg agagagatat catcttgcaaggatgcctgc 1800 tttacaaaca tccttgaaac aacaatccag aaaaaaaaag gtgttactgtctttgctcag 1860 aagacacaca gatacgtgac agaaccatgg agaattgcct cccaacgctgttcagccaga 1920 gccttccacc ctttctgcag gacagtctca acgttccacc-attaaatacttcttctatca 1980 catcccgctt ctttatgcct aaccaaggtt ctaggtcccg atcgactgtgtctggcagca 2040 ctccactgcc aaacccagaa taaggcagcg ctcaggatcc cgaaggggcatggctgggga 2100 tcagaacttc tgggtttgag tgaggagtgg gtccaccctc ttgaatttcaaaggaggaag 2160 aggctggatg tgaaggtact gggggaggga aagtgtcagt tccgaactcttaggtcaatg 2220 agggaggaga ctggtaaggt cccagctccc gaggtactga tgtgggaatggcctaagaat 2280 ctcatatcct caggaagaag gtgctggaat cctgaggggt agagttctgggtatatttgt 2340 ggcttaaggc tctttggccc ctgaaggcag aggctggaac cattaggtccagggtttggg 2400 gtgatagtaa tgggatctct tgattcctca agagtctgag gatcgagggttgcccattct 2460 tccatcttgc cacctaatcc ttactccact tgagggtatc accagcccttctagctccat 2520 gaaggtcccc tgggcaagca caatctgagc atgaaagatg ccccagaggccttgggtgtc 2580 atccactcat catccagcat cacactctga gggtgtggcc agcaccatgacgtcatgttg 2640 ctgtgactat ccctgcagcg tgcctctcca gccacctgcc aaccgtagagctgcccatcc 2700 tcctctggtg ggagtggcct gcatggtgcc aggctgaggc ctagtgtcagacagggagcc 2760 tggaatcata gggatccagg actcaaaagt gctagagaat ggccatatgtcaccatccat 2820 gaaatctcaa gggcttctgg gtggagggca cagggacctg aacttatggtttcccaagtc 2880 tattgctctc ccaagtgagt ctcccagata cgaggcactg tgccagcatcagccttatct 2940 ccaccacatc ttgtaaaagg actacccagg gccctgatga acaccatggtgtgtacagga 3000 gtagggggtg gaggcacgga ctcctgtgag gtcacagcca agggagcatcatcatgggtg 3060 gggaggaggc aatggacagg cttgagaacg gggatgtggt tgtatttggttttctttggt 3120 tagataaagt gctgggtata ggattgagag tggagtatga agaccagttaggatggagga 3180 tcagattgga gttgggttag ataaagtgct gggtatagga ttgagagtggagtatgaaga 3240 ccagttagga tggaggatca gattggagtt gggttagaga tggggtaaaattgtgctccg 3300 gatgagtttg ggattgacac tgtggaggtg gtttgggatg gcatggctttgggatggaaa 3360 tagatttgtt ttgatgttgg ctcagacatc cttggggatt gaactggggatgaagctggg 3420 tttgattttg gaggtagaag acgtggaagt agctgtcaga tttgacagtggccatgagtt 3480 ttgtttgatg gggaatcaaa caatggggga agacataagg gttggcttgttaggttaagt 3540 tgcgttgggt tgatggggtc ggggctgtgt ataatgcagt tggattggtttgtattaaat 3600 tgggttgggt caggttttgg ttgaggatga gttgaggata tgcttggggacaccggatcc 3660 atgaggtect cactggagtg gagacaaact tcctttccag gatgaatccagggaagcctt 3720 aattcacgtg taggggaggt caggccactg gctaagtata tccttccactccagctctaa 3780 gatggtctta aattgtgatt atctatatcc acttctgtct ccctcactgtgcttggagtt 3840 tacctgatca ctcaactaga aacaggggaa gattttatca aattcttttttttttttttt 3900 tttttttgag acagagtctc actctgttgc ccaggctgga gtgcagtggcgcagtctcgg 3960 ctcactgcaa cctctgcctc ccaggttcaa gtgattctcc tgcctcagcctcctgagttg 4020 ctgggattac aggcatgcag caccatgccc agctaatttt tgtatttttagtagagatgg 4080 ggtttcacca atgtttgcca ggctggcctc gaactcctga cctggtgatccacctgcctc 4140 agcctcccaa agtgctggga ttacaggcgt cagccaccgc gcccagccacttttgtcaaa 4200 ttcttgagac acagctcggg ctggatcaag tgagctactc tggttttattgaacagctga 4260 aataaccaac tttttggaaa ttgatgaaat cttacggagt taacagtggaggtaccaggg 4320 ctcttaagag ttcccgattc tcttctgaga ctacaaattg tgattttgcatgccacctta 4380 atcttttttt tttttttttt aaatcgaggt ttcagtctca ttctatttcccaggctggag 4440 ttcaatagcg tgatcacagc tcactgtagc cttgaactcc tggccttaagagattctcct 4500 gcttcggtct cccaatagct aagactacag tagtccacca ccatatccagataattttta 4560 aattttttgg ggggccgggc acagtggctc acgcctgtaa tcccaacaccatgggaggct 4620 gagatgggtg gatcacgagg tcaggagttt gagaccagcc tgaccaacatggtgaaactc 4680 tgtctctact aaaaaaaaaa aaaatagaaa aattagccgg gcgtggtggcacacggcacc 4740 tgtaatccca gctactgagg aggctgaggc aggagaatca cttgaacccagaaggcagag 4800 gttgcaatga gccgagattg cgccactgca ctccagcctg ggtgacagagtgagactctg 4860 tctcaaaaaa aaaaaatttt tttttttttt ttgtagagat ggatcttgctttgtttctct 4920 ggttggcctt gaactcctgg cttcaagtga tcctcctacc ttggcctcggaaagtgttgg 4980 gattacaggc gtgagccacc atgactgacc tgtcgttaat cttgaggtacataaacctgg 5040 ctcctaaagg ctaaaggcta aatatttgtt ggagaagggg cattggattttgcatgagga 5100 tgattctgac ctgggagggc aggtcagcag gcatctctgt tgcacagatagagtgtacag 5160 gtctggagaa caaggagtgg ggggttattg gaattccaca ttgtttgctgcacgttggat 5220 tttgaaatgc tagggaactt tgggagactc atatttctgg gctagaggatctgtggacca 5280 caagatcttt ttatgatgac agtagcaatg tatctgtgga gctggattctgggttgggag 5340 tgcaaggaaa agaatgtact aaatgccaag acatctattt caggagcatgaggaataaaa 5400 gttctagttt ctggtctcag agtggtgcat ggatcaggga gtctcacaatctcctgagtg 5460 ctggtgtctt agggcacact gggtcttgga gtgcaaagga tctaggcacgtgaggctttg 5520 tatgaagaat cggggatcgt acccaccccc tgtttctgtt tcatcctgggcatgtctcct 5580 ctgcctttgt cccctagatg aagtctccat gagctacaag ggcctggtgcatccagggtg 5640 atctagtaat tgcagaacag caagtgctag ctctccctcc ccttccacagctctgggtgt 5700 gggagggggt tgtccagcct ccagcagcat ggggagggcc ttggtcagcctctgggtgcc 5760 agcagggcag gggcggagtc ctggggaatg aaggttttat agggctcctgggggaggctc 5820 cccagcccca agctt 5835 CEA TRE SEQ ID NO: aagctttttagtgctttaga cagtgagctg gtctgtctaa cccaagtgac ctgggctcca 60 tactcagccccagaagtgaa gggtgaagct gggtggagcc aaaccaggca agcctaccct 120 cagggctcccagtggcctga gaaccattgg acccaggacc cattacttct agggtaagga 180 aggtacaaacaccagatcca accatggtct ggggggacag ctgtcaaatg cctaaaaata 240 tacctgggagaggagcaggc aaactatcac tgccccaggt tctctgaaca gaaacagagg 300 ggcaacccaaagtccaaatc caggtgagca ggtgcaccaa atgcccagag atatgacgag 360 gcaagaagtgaaggaaccac ccctgcatca aatgttttgc atgggaagga gaagggggtt 420 gctcatgttcccaatccagg agaatgcatt tgggatctgc cttcttctca ctccttggtt 480 agcaagactaagcaaccagg actctggatt tggggaaaga cgtttatttg tggaggccag 540 tgatgacaatcccacgaggg cctaggtgaa gagggcagga aggctcgaga cactggggac 600 tgagtgaaaaccacacccat gatctgcacc acccatggat gctccttcat tgctcacctt 660 tctgttgatatcagatggcc ccattttctg taccttcaca gaaggacaca ggctagggtc 720 tgtgcatggccttcatcccc ggggccatgt gaggacagca ggtgggaaag atcatgggtc 780 ctcctgggtcctgcagggcc agaacattca tcacccatac tgacctccta gatgggaatg 840 gcttccctggggctgggcca acggggcctg ggcaggggag aaaggacgtc aggggacagg 900 gaggaagggtcatcgagacc cagcctggaa ggttcttgtc tctgaccatc caggatttac 960 ttccctgcatctacctttgg tcattttccc tcagcaatga ccagctctgc ttcctgatct 1020 cagcctcccaccctggacac agcaccccag tccctggccc ggctgcatcc acccaatacc 1080 ctgataacccaggacccatt acttctaggg taaggagggt ccaggagaca gaagctgagg 1140 aaaggtctgaagaagtcaca tctgtcctgg ccagagggga aaaaccatca gatgctgaac 1200 caggagaatgttgacccagg aaagggaccg aggacccaag aaaggagtca gaccaccagg 1260 gtttgcctgagaggaaggat caaggccccg agggaaagca gggctggctg catgtgcagg 1320 acactggtggggcatatgtg tcttagattc tccctgaatt cagtgtccct gccatggcca 1380 gactctctactcaggcctgg acatgctgaa ataggacaat ggccttgtcc tctctcccca 1440 ccatttggcaagagacataa aggacattcc aggacatgcc ttcctgggag gtccaggttc 1500 tctgtctcacacctcaggga ctgtagttac tgcatcagcc atggtaggtg ctgatctcac 1560 ccagcctgtccaggcccttc cactctccac tttgtgacca tgtccaggac cacccctcag 1620 atcctgagcctgcaaatacc cccttgctgg gtgggtggat tcagtaaaca gtgagctcct 1680atccagcccc-cagagccacc tctgtcacct tcctgctggg catcatccca ccttcacaag 1740cactaaagag catggggaga cctggctagc tgggtttctg catcacaaag aaaataatcc 1800cccaggttcg gattcccagg gctctgtatg tggagctgac agacctgagg ccaggagata 1860gcagaggtca gccctaggga gggtgggtca tccacccagg ggacaggggt gcaccagcct 1920tgctactgaa agggcctccc caggacagcg ccatcagccc tgcctgagag ctttgctaaa 1980cagcagtcag aggaggccat ggcagtggct gagctcctgc tccaggcccc aacagaccag 2040accaacagca caatgcagtc cttccccaac gtcacaggtc accaaaggga aactgaggtg 2100ctacctaacc ttagagccat caggggagat aacagcccaa tttcccaaac aggccagttt 2160caatcccatg acaatgacct ctctgctctc attcttccca aaataggacg ctgattctcc 2220cccaccatgg atttctccct tgtcccggga gccttttctg ccccctatga tctgggcact 2280cctgacacac acctcctctc tggtgacata tcagggtccc tcactgtcaa gcagtccaga 2340aaggacagaa ccttggacag cgcccatctc agcttcaccc ttcctccttc acagggttca 2400gggcaaagaa taaatggcag aggccagtga gcccagagat ggtgacaggc agtgacccag 2460gggcagatgc ctggagcagg agctggcggg gccacaggga gaaggtgatg caggaaggga 2520aacccagaaa tgggcaggaa aggaggacac aggctctgtg gggctgcagc ccagggttgg 2580actatgagtg tgaagccatc tcagcaagta aggccaggtc ccatgaacaa gagtgggagc 2640acgtggcttc ctgctctgta tatggggtgg gggattccat gccccataga accagatggc 2700cggggttcag atggagaagg agcaggacag gggatcccca ggataggagg accccagtgt 2760ccccacccag gcaggtgact gatgaatggg catgcagggt cctcctgggc tgggctctcc 2820ctttgtccct caggattcct tgaaggaaca tccggaagcc gaccacatct acctggtggg 2880ttctggggag tccatgtaaa gccaggagct tgtgttgcta ggaggggtca tggcatgtgc 2940tgggggcacc aaagagagaa acctgagggc aggcaggacc tggtctgagg aggcatggga 3000gcccagatgg ggagatggat gtcaggaaag gctgccccat cagggagggt gatagcaatg 3060gggggtctgt gggagtgggc acgtgggatt ccctgggctc tgccaagttc cctcccatag 3120tcacaacctg gggacactgc ccatgaaggg gcgcctttgc ccagccagat gctgctggtt 3180ctgcccatcc actaccctct ctgctccagc cactctgggt ctttctccag atgccctgga 3240caaccctggc ctgggcctgt cccctgagag gtgttgggag aagctgagtc tctggggaca 3300ctctcatcag agtctgaaag gcacatcagg aaacatccct ggtctccagg actaggcaat 3360gaggaaaggg ccccagctcc tccctttccc actgagaggg tcgaccctgg gtggccacag 3420tgacttctgc gtctgtccca gtcaccctga aaccacaaca aaaccccagc cccagaccct 3480gcaggtacaa tacatgtggg gacagtctgt acccagggga agccagttct ctcttcctag 3540gagaccgggc ctcagggctg tgcccggggc aggcgggggc agcacgtgcc tgtccttgag 3600aactcgggac cttaagggtc tctgctctgt gaggcacagc aaggatcctt ctgtccagag 3660atgaaagcag ctcctgcccc tcctctgacc tcttcctcct tcccaaatct caaccaacaa 3720ataggtgttt caaatctcat catcaaatct tcatccatcc acatgagaaa gcttaaaacc 3780caatggattg acaacatcaa gagttggaac aagtggacat ggagatgtta cttgtggaaa 3840tttagatgtg ttcagctatc gggcaggaga atctgtgtca aattccagca tggttcagaa 3900gaatcaaaaa gtgtcacagt ccaaatgtgc aacagtgcag gggataaaac tgtggtgcat 3960tcaaactgag ggatattttg gaacatgaga aaggaaggga ttgctgctgc acagaacatg 4020gatgatctca cacatagagt tgaaagaaag gagtcaatcg cagaatagaa aatgatcact 4080aattccacct ctataaagtt tccaagagga aaacccaatt ctgctgctag agatcagaat 4140ggaggtgacc tgtgccttgc aatggctgtg agggtcacgg gagtgtcact tagtgcaggc 4200aatgtgccgt atcttaatct gggcagggct ttcatgagca cataggaatg cagacattac 4260tgctgtgttc attttacttc accggaaaag aagaataaaa tcagccgggc gcggtggctc 4320acgcctgtaa tcccagcact ttagaaggct gaggtgggca gattacttga ggtcaggagt 4380tcaagaccac cctggccaat atggtgaaac cccggctcta ctaaaaatac aaaaattagc 4440tgggcatggt ggtgcgcgcc tgtaatccca gctactcggg aggctgaggc tggacaattg 4500cttggaccca ggaagcagag gttgcagtga gccaagattg tgccactgca ctccagcttg 4560ggcaacagag ccagactctg taaaaaaaaa aaaaaaaaaa aaaaaaagaa agaaagaaaa 4620agaaaagaaa gtataaaatc tctttgggtt aacaaaaaaa gatccacaaa acaaacacca 4680gctcttatca aacttacaca actctgccag agaacaggaa acacaaatac tcattaactc 4740acttttgtgg caataaaacc ttcatgtcaa aaggagacca ggacacaatg aggaagtaaa 4800actgcaggcc ctacttgggt gcagagaggg aaaatccaca aataaaacat taccagaagg 4860agctaagatt tactgcattg agttcattcc ccaggtatgc aaggtgattt taacacctga 4920aaatcaatca ttgcctttac tacatagaca gattagctag aaaaaaatta caactagcag 4980cacagaagca atttggcctt cctaaaattc cacatcatat catcatgatg gagacagtgc 5040agacgccaat gacaataaaa agagggacct ccgtcacccg gtaaacatgt ccacacagct 5100ccagcaagca-cccgtcttcc cagtgaatca ctctaacctc ccctttaatc agccccaggc 5160aaggctgcct gcgatggcca cacaggctcc aacccgtggg cctcaacctc ccgcagaggc 5220tctcctttgg ccaccccatg gggagagcat gaggacaggg cagagccctc tgatgcccac 5280acatggcagg agctgacgcc agagccatgg gggctggaga gcagagctgc tggggtcaga 5340gcttcctgag gacacccagg cctaagggaa ggcagctccc tggatggggg caaccaggct 5400ccgggctcca acctcagagc ccgcatggga ggagccagca ctctaggcct ttcctagggt 5460gactctgagg ggaccctgac acgacaggat cgctgaatgc acccgagatg aaggggccac 5520cacgggaccc tgctctcgtg gcagatcagg agagagtggg acaccatgcc aggcccccat 5580ggcatggctg cgactgaccc aggccactcc cctgcatgca tcagcctcgg taagtcacat 5640gaccaagccc aggaccaatg tggaaggaag gaaacagcat cccctttagt gatggaaccc 5700aaggtcagtg caaagagagg ccatgagcag ttaggaaggg tggtccaacc tacagcacaa 5760accatcatct atcataagta gaagccctgc tccatgaccc ctgcatttaa ataaacgttt 5820gttaaatgag tcaaattccc tcaccatgag agcccacctg tgtgtaggcc catcacacac 5880acaaacacac acacacacac acacacacac acacacacac acagggaaag tgcaggatcc 5940tggacagcac caggcaggct tcacaggcag agcaaacagc gtgaatgacc catgcagtgc 6000cctgggcccc atcagctcag agaccctgtg agggctgaga tggggctagg caggggagag 6060acttagagag ggtggggcct ccagggaggg ggctgcaggg agctgggtac tgccctccag 6120ggagggggct gcagggagct gggtactgcc ctccagggag ggggctgcag ggagctgggt 6180actgccctcc agggaggggg ctgcagggag ctgggtactg ccctccaggg agggggctgc 6240agggagctgg gtactgccct ccagggaggc aggagcactg ttcccaacag agagcacatc 6300ttcctgcagc agctgcacag acacaggagc ccccatgact gccctgggcc agggtgtgga 6360ttccaaattt cgtgccccat tgggtgggac ggaggttgac cgtgacatcc aaggggcatc 6420tgtgattcca aacttaaact actgtgccta caaaatagga aataacccta ctttttctac 6480tatctcaaat tccctaagca caagctagca ccctttaaat caggaagttc agtcactcct 6540ggggtcctcc catgccccca gtctgacttg caggtgcaca gggtggctga catctgtcct 6600tgctcctcct cttggctcaa ctgccgcccc tcctgggggt gactgatggt caggacaagg 6660gatcctagag ctggccccat gattgacagg aaggcaggac ttggcctcca ttctgaagac 6720taggggtgtc aagagagctg ggcatcccac agagctgcac aagatgacgc ggacagaggg 6780tgacacaggg ctcagggctt cagacgggtc gggaggctca gctgagagtt cagggacaga 6840cctgaggagc ctcagtggga aaagaagcac tgaagtggga agttctggaa tgttctggac 6900aagcctgagt gctctaagga aatgctccca ccccgatgta gcctgcagca ctggacggtc 6960tgtgtacctc cccgctgccc atcctctcac agcccccgcc tctagggaca caactcctgc 7020cctaacatgc atctttcctg tctcattcca cacaaaaggg cctctggggt ccctgttctg 7080cattgcaagg agtggaggtc acgttcccac agaccaccca gcaacagggt cctatggagg 7140tgcggtcagg aggatcacac gtccccccat gcccagggga ctgactctgg gggtgatgga 7200ttggcctgga ggccactggt cccctctgtc cctgagggga atctgcaccc tggaggctgc 7260cacatccctc ctgattcttt cagctgaggg cccttcttga aatcccaggg aggactcaac 7320ccccactggg aaaggcccag tgtggacggt tccacagcag cccagctaag gcccttggac 7380acagatcctg agtgagagaa cctttaggga cacaggtgca cggccatgtc cccagtgccc 7440acacagagca ggggcatctg gaccctgagt gtgtagctcc cgcgactgaa cccagccctt 7500ccccaatgac gtgacccctg gggtggctcc aggtctccag tccatgccac caaaatctcc 7560agattgaggg tcctcccttg agtccctgat gcctgtccag gagctgcccc ctgagcaaat 7620ctagagtgca gagggctggg attgtggcag taaaagcagc cacatttgtc tcaggaagga 7680aagggaggac atgagctcca ggaagggcga tggcgtcctc tagtgggcgc ctcctgttaa 7740tgagcaaaaa ggggccagga gagttgagag atcagggctg gccttggact aaggctcaga 7800tggagaggac tgaggtgcaa agagggggct gaagtagggg agtggtcggg agagatggga 7860ggagcaggta aggggaagcc ccagggaggc cgggggaggg tacagcagag ctctccactc 7920ctcagcattg acatttgggg tggtcgtgct agtggggttc tgtaagttgt agggtgttca 7980gcaccatctg gggactctac ccactaaatg ccagcaggac tccctcccca agctctaaca 8040accaacaatg tctccagact ttccaaatgt cccctggaga gcaaaattgc ttctggcaga 8100atcactgatc tacgtcagtc tccaaaagtg actcatcagc gaaatccttc acctcttggg 8160agaagaatca caagtgtgag aggggtagaa actgcagact tcaaaatctt tccaaaagag 8220ttttacttaa tcagcagttt gatgtcccag gagaagatac atttagagtg tttagagttg 8280atgccacatg gctgcctgta cctcacagca ggagcagagt gggttttcca agggcctgta 8340accacaactg gaatgacact cactgggtta cattacaaag tggaatgtgg ggaattctgt 8400agactttggg aagggaaatg tatgacgtca gcccacagcc taaggcagtg gacagtccac 8460tttgaggctc tcaccatcta ggagacatct cagccatgaa catagccaca tctgtcatta 8520gaaaacatgt-tttattaaga ggaaaaatct aggctagaag tgctttatgc tcttttttct 8580ctttatgttc aaattcatat acttttagat cattccttaa agaagaatct atccccctaa 8640gtaaatgtta tcactgactg gatagtgttg gtgtctcact cccaacccct gtgtggtgac 8700agtgccctgc ttccccagcc ctgggccctc tctgattcct gagagctttg ggtgctcctt 8760cattaggagg aagagaggaa gggtgttttt aatattctca ccattcaccc atccacctct 8820tagacactgg gaagaatcag ttgcccactc ttggatttga tcctcgaatt aatgacctct 8880atttctgtcc cttgtccatt tcaacaatgt gacaggccta agaggtgcct tctccatgtg 8940atttttgagg agaaggttct caagataagt tttctcacac ctctttgaat tacctccacc 9000tgtgtcccca tcaccattac cagcagcatt tggacccttt ttctgttagt cagatgcttt 9060ccacctcttg agggtgtata ctgtatgctc tctacacagg aatatgcaga ggaaatagaa 9120aaagggaaat cgcattacta ttcagagaga agaagacctt tatgtgaatg aatgagagtc 9180taaaatccta agagagccca tataaaatta ttaccagtgc taaaactaca aaagttacac 9240taacagtaaa ctagaataat aaaacatgca tcacagttgc tggtaaagct aaatcagata 9300tttttttctt agaaaaagca ttccatgtgt gttgcagtga tgacaggagt gcccttcagt 9360caatatgctg cctgtaattt ttgttccctg gcagaatgta ttgtcttttc tccctttaaa 9420tcttaaatgc aaaactaaag gcagctcctg ggccccctcc ccaaagtcag ctgcctgcaa 9480ccagccccac gaagagcaga ggcctgagct tccctggtca aaataggggg ctagggagct 9540taaccttgct cgataaagct gtgttcccag aatgtcgctc ctgttcccag gggcaccagc 9600ctggagggtg gtgagcctca ctggtggcct gatgcttacc ttgtgccctc acaccagtgg 9660tcactggaac cttgaacact tggctgtcgc ccggatctgc agatgtcaag aacttctgga 9720agtcaaatta ctgcccactt ctccagggca gatacctgtg aacatccaaa accatgccac 9780agaaccctgc ctggggtcta caacacatat ggactgtgag caccaagtcc agccctgaat 9840ctgtgaccac ctgccaagat gcccctaact gggatccacc aatcactgca catggcaggc 9900agcgaggctt ggaggtgctt cgccacaagg cagccccaat ttgctgggag tttcttggca 9960cctggtagtg gtgaggagcc ttgggaccct caggattact ccccttaagc atagtgggga 10020cccttctgca tccccagcag gtgccccgct cttcagagcc tctctctctg aggtttaccc 10080agacccctgc accaatgaga ccatgctgaa gcctcagaga gagagatgga gctttgacca 10140ggagccgctc ttccttgagg gccagggcag ggaaagcagg aggcagcacc aggagtggga 10200acaccagtgt ctaagcccct gatgagaaca gggtggtctc tcccatatgc ccataccagg 10260cctgtgaaca gaatcctcct tctgcagtga caatgtctga gaggacgaca tgtttcccag 10320cctaacgtgc agccatgccc atctacccac tgcctactgc aggacagcac caacccagga 10380gctgggaagc tgggagaaga catggaatac ccatggcttc tcaccttcct ccagtccagt 10440gggcaccatt tatgcctagg acacccacct gccggcccca ggctcttaag agttaggtca 10500cctaggtgcc tctgggaggc cgaggcagga gaattgcttg aacccgggag gcagaggttg 10560cagtgagccg agatcacacc actgcactcc agcctgggtg acagaatgag actctgtctc 10620aaaaaaaaag agaaagatag catcagtggc taccaagggc taggggcagg ggaaggtgga 10680gagttaatga ttaatagtat gaagtttcta tgtgagatga tgaaaatgtt ctggaaaaaa 10740aaatatagtg gtgaggatgt agaatattgt gaatataatt aacggcattt aattgtacac 10800ttaacatgat taatgtggca tattttatct tatgtatttg actacatcca agaaacactg 10860ggagagggaa agcccaccat gtaaaataca cccaccctaa tcagatagtc ctcattgtac 10920ccaggtacag gcccctcatg acctgcacag gaataactaa ggatttaagg acatgaggct 10980tcccagccaa ctgcaggtgc acaacataaa tgtatctgca aacagactga gagtaaagct 11040gggggcacaa acctcagcac tgccaggaca cacacccttc tcgtggattc tgactttatc 11100tgacccggcc cactgtccag atcttgttgt gggattggga caagggaggt cataaagcct 11160gtccccaggg cactctgtgt gagcacacga gacctcccca cccccccacc gttaggtctc 11220cacacataga tctgaccatt aggcattgtg aggaggactc tagcgcgggc tcagggatca 11280caccagagaa tcaggtacag agaggaagac ggggctcgag gagctgatgg atgacacaga 11340gcagggttcc tgcagtccac aggtccagct caccctggtg taggtgcccc atccccctga 11400tccaggcatc cctgacacag ctccctcccg gagcctcctc ccaggtgaca catcagggtc 11460cctcactcaa gctgtccaga gagggcagca ccttggacag cgcccacccc acttcactct 11520tcctccctca cagggctcag ggctcagggc tcaagtctca gaacaaatgg cagaggccag 11580tgagcccaga gatggtgaca gggcaatgat ccaggggcag ctgcctgaaa cgggagcagg 11640tgaagccaca gatgggagaa gatggttcag gaagaaaaat ccaggaatgg gcaggagagg 11700agaggaggac acaggctctg tggggctgca gcccaggatg ggactaagtg tgaagacatc 11760tcagcaggtg aggccaggtc ccatgaacag agaagcagct cccacctccc ctgatgcacg 11820gacacacaga gtgtgtggtg ctgtgccccc agagtcgggc tctcctgttc tggtccccag 11880ggagtgagaa gtgaggttga cttgtccctg ctcctctctg ctaccccaac attcaccttc 11940tcctcatgcc-cctctctctc aaatatgatt tggatctatg tccccgccca aatctcatgt 12000caaattgtaa accccaatgt tggaggtggg gccttgtgag aagtgattgg ataatgcggg 12060tggattttct gctttgatgc tgtttctgtg atagagatct cacatgatct ggttgtttaa 12120aagtgtgtag cacctctccc ctctctctct ctctctctta ctcatgctct gccatgtaag 12180acgttcctgt ttccccttca ccgtccagaa tgattgtaag ttttctgagg cctccccagg 12240agcagaagcc actatgcttc ctgtacaact gcagaatgat gagcgaatta aacctctttt 12300ctttataaat tacccagtct caggtatttc tttatagcaa tgcgaggaca gactaataca 12360atcttctact cccagatccc cgcacacgct tagccccaga catcactgcc cctgggagca 12420tgcacagcgc agcctcctgc cgacaaaagc aaagtcacaa aaggtgacaa aaatctgcat 12480ttggggacat ctgattgtga aagagggagg acagtacact tgtagccaca gagactgggg 12540ctcaccgagc tgaaacctgg tagcactttg gcataacatg tgcatgaccc gtgttcaatg 12600tctagagatc agtgttgagt aaaacagcct ggtctggggc cgctgctgtc cccacttccc 12660tcctgtccac cagagggcgg cagagttcct cccaccctgg agcctcccca ggggctgctg 12720acctccctca gccgggccca cagcccagca gggtccaccc tcacccgggt cacctcggcc 12780cacgtcctcc tcgccctccg agctcctcac acggactctg tcagctcctc cctgcagcct 12840atcggccgcc cacctgaggc ttgtcggccg cccacttgag gcctgtcggc tgccctctgc 12900aggcagctcc tgtcccctac accccctcct tccccgggct cagctgaaag ggcgtctccc 12960agggcagctc cctgtgatct ccaggacagc tcagtctctc acaggctccg acgcccccta 13020tgctgtcacc tcacagccct gtcattacca ttaactcctc agtcccatga agttcactga 13080gcgcctgtct cccggttaca ggaaaactct gtgacaggga ccacgtctgt cctgctctct 13140gtggaatccc agggcccagc ccagtgcctg acacggaaca gatgctccat aaatactggt 13200taaatctgtg ggagatctct aaaaagaagc atatcacctc cgtgtggccc ccagcagtca 13260gagtctgttc catgtggaca caggggcact ggcaccagca tgggaggagg ccagcaagtg 13320cccgccgctg ccccaggaat gaggcctcaa cccccagagc ttcagaaggg aggacagagg 13380cctgcaggga atagatcctc cggcctgacc ctgcagccta atccagagtt cagggtcagc 13440tcacaccacg tcgaccctgg tcagcatccc tagggcagtt ccagacaagg ccggaggtct 13500cctcttgccc tccagggggt gacattgcac acagacatca ctcaggaaac ggattcccct 13560ggacacgaac ctggctttgc taaggaagtg gaggtggagc ctggtttcca tcccttgctc 13620caacagaccc ttctgatctc tcccacatac ctgctctgtt cctttctggg tcctatgagg 13680accctgttct gccaggggtc cctgtgcaac tccagactcc ctcctggtac caccatgggg 13740aaggtggggt gatcacagga cagtcagcct cgcagagaca cagaccaccc aggactgtca 13800gggagaacat ggacaggccc tgagccgcag ctcagccaac agacacggag agggagggtc 13860cccctggagc cttccccaag gacagcagag cccagagtca cccacctccc tccaccacag 13920tcctctcttt ccaggacaca caagacacct ccccctccac atgcaggatc tggggactcc 13980tgagacctct gggcctgggt ctccatccct gggtcagtgg cggggttggt ggtactggag 14040acagagggct ggtccctccc cagccaccac ccagtgagcc tttttctagc ccccagagcc 14100acctctgtca ccttcctgtt gggcatcatc ccaccttccc agagccctgg agagcatggg 14160gagacccggg accctgctgg gtttctctgt cacaaaggaa aataatcccc ctggtgtgac 14220agacccaagg acagaacaca gcagaggtca gcactgggga agacaggttg tcctcccagg 14280ggatgggggt ccatccacct tgccgaaaag atttgtctga ggaactgaaa atagaaggga 14340aaaaagagga gggacaaaag aggcagaaat gagaggggag gggacagagg acacctgaat 14400aaagaccaca cccatgaccc acgtgatgct gagaagtact cctgccctag gaagagactc 14460     ⇓ transcription start site agggcagagg gaggaaggac agcagaccagacagtcacag cagccttgac aaaacgttcc 14520 tggaactcaa gctcttctcc acagaggaggacagagcaga cagcagagac catggagtct 14580 ccctcggccc ctccccacag atggtgcatcccctggcaga ggctcctgct cacaggtgaa 14640 gggaggacaa cctgggagag ggtgggaggagggagctggg gtctcctggg taggacaggg 14700 ctgtgagacg gacagagggc tcctgttggagcctgaatag ggaagaggac atcagagagg 14760 gacaggagtc acaccagaaa aatcaaattgaactggaatt ggaaaggggc aggaaaacct 14820 caagagttct attttcctag ttaattgtcactggccacta cgtttttaaa aatcataata 14880 actgcatcag atgacacttt aaataaaaacataaccaggg catgaaacac tgtcctcatc 14940 cgcctaccgc ggacattgga aaataagccccaggctgtgg agggccctgg gaaccctcat 15000 gaactcatcc acaggaatct gcagcctgtcccaggcactg gggtgcaacc aagatc 15056 Mucin-TRE SEQ ID NO:_(—) cgagcggcccctcagcttcg gcgcccagcc ccgcaaggct cccggtgacc actagagggc 60 gggaggagctcctggccagt ggtggagagt ggcaaggaag gaccctaggg ttcatcggag 120 cccaggtttactcccttaag tggaaatttc ttcccccact cctccttggc tttctccaag 180 gagggaacccaggctgctgg aaagtccggc tggggcgggg actgtgggtt caggggagaa 240 cggggtgtggaacgggacag ggagcggtta gaagggtggg gctattccgg gaagtggtgg 300 ggggagggagcccaaaacta gcacctagtc cactcattat ccagccctct tatttctcgg 360 ccgctctgcttcagtggacc cggggagggc ggggaagtgg agtgggagac ctaggggtgg 420 gcttcccgaccttgctgtac aggacctcga cctagctggc tttgttcccc atccccacgt 480 tagttgttgccctgaggcta aaactagagc ccaggggccc caagttccag actgcccctc 540 ccccctcccccggagccagg gagtggttgg tgaaaggggg aggccagctg gagaacaaac 600 gggtagtcagggggttgagc gattagagcc cttgtaccct acccaggaat ggttggggag 660 gaggaggaagaggtaggagg taggggaggg ggcggggttt tgtcacctgt cacctgctcg 720 ctgtgcctagggcgggcggg cggggagtgg ggggaccggt ataaagcggt aggcgcctgt 780 gcccgctccacctctcaagc agccagcgcc tgcctgaatc tgttctgccc cctccccacc 840 catttcaccaccaccatg 858 αFP-TRE SEQ ID NO:_(—) gaattcttag aaatatgggg gtaggggtggtggtggtaat tctgttttca ccccataggt 60 gagataagca ttgggttaaa tgtgctttcacacacacatc acatttcata agaattaagg 120 aacagactat gggctggagg actttgaggatgtctgtctc ataacacttg ggttgtatct 180 gttctatggg gcttgtttta agcttggcaacttgcaacag ggttcactga ctttctcccc 240 aagcccaagg tactgtcctc ttttcatatctgttttgggg cctctggggc ttgaatatct 300 gagaaaatat aaacatttca ataatgttctgtggtgagat gagtatgaga gatgtgtcat 360 tcatttgtat caatgaatga atgaggacaattagtgtata aatccttagt acaacaatct 420 gagggtaggg gtggtactat tcaatttctatttataaaga tacttatttc tatttattta 480 tgcttgtgac aaatgttttg ttcgggaccacaggaatcac aaagatgagt ctttgaattt 540 aagaagttaa tggtccagga ataattacatagcttacaaa tgactatgat ataccatcaa 600 acaagaggtt ccatgagaaa ataatctgaaaggtttaata agttgtcaaa ggtgagaggg 660 ctcttctcta gctagagact aatcagaaatacattcaggg ataattattt gaatagacct 720 taagggttgg gtacattttg ttcaagcattgatggagaag gagagtgaat atttgaaaac 780 attttcaact aaccaaccac ccaatccaacaaacaaaaaa tgaaaagaat ctcagaaaca 840 gtgagataag agaaggaatt ttctcacaacccacacgtat agctcaactg ctctgaagaa 900 gtatatatct aatatttaac actaacatcatgctaataat gataataatt actgtcattt 960 tttaatgtct ataagtacca ggcatttagaagatattatt ccatttatat atcaaaataa 1020 acttgagggg atagatcatt ttcatgatatatgagaaaaa ttaaaaacag attgaattat 1080 ttgcctgtca tacagctaat aattgaccataagacaatta gatttaaatt agttttgaat 1140 ctttctaata ccaaagttca gtttactgttccatgttgct tctgagtggc ttcacagact 1200 tatgaaaaag taaacggaat cagaattacatcaatgcaaa agcattgctg tgaactctgt 1260 acttaggact aaactttgag caataacacacatagattga ggattgtttg ctgttagcat 1320 acaaactctg gttcaaagct cctctttattgcttgtcttg gaaaatttgc tgttcttcat 1380 ggtttctctt ttcactgcta tctatttttctcaaccactc acatggctac aataactgtc 1440 tgcaagctta tgattcccaa atatctatctctagcctcaa tcttgttcca gaagataaaa 1500 agtagtattc aaatgcacat caacgtctccacttggaggg cttaaagacg tttcaacata 1560 caaaccgggg agttttgcct ggaatgtttcctaaaatgtg tcctgtagca catagggtcc 1620 tcttgttcct taaaatctaa ttacttttagcccagtgctc atcccaccta tggggagatg 1680 agagtgaaaa gggagcctga ttaataattacactaagtca ataggcatag agccaggact 1740 gtttgggtaa actggtcact ttatcttaaactaaatatat ccaaaactga acatgtactt 1800 agttactaag tctttgactt tatctcattcataccactca gctttatcca ggccacttat 1860 ttgacagtat tattgcgaaa acttcctaactggtctcctt atcatagtct tatccccttt 1920 tgaaacaaaa gagacagttt caaaatacaaatatgatttt tattagctcc cttttgttgt 1980 ctataatagt cccagaagga gttataaactccatttaaaa agtctttgag atgtggccct 2040 tgccaacttt gccaggaatt cccaatatctagtattttct actattaaac tttgtgcctc 2100 ttcaaaactg cattttctct cattccctaagtgtgcattg ttttccctta ccggttggtt 2160 tttccaccac cttttacatt ttcctggaacactataccct ccctcttcat ttggcccacc 2220 tctaattttc tttcagatct ccatgaagatgttacttcct ccaggaagcc ttatctgacc 2280 cctccaaaga tgtcatgagt tcctcttttcattctactaa tcacagcatc catcacacca 2340 tgttgtgatt actgatacta ttgtctgtttctctgattag gcagtaagct caacaagagc 2400 tacatggtgc ctgtctcttg ttgctgattattcccatcca aaaacagtgc ctggaatgca 2460 gacttaacat tttattgaat gaataaataaaaccccatct atcgagtgct actttgtgca 2520 agacccggtt ctgaggcatt tatatttattgatttattta attctcattt aaccatgaag 2580 gaggtactat cactatcctt attttatagttgataaagat aaagcccaga gaaatgaatt 2640 aactcaccca aagtcatgta gctaagtgacagggcaaaaa ttcaaaccag ttccccaact 2700 ttacgtgatt aatactgtgc tatactgcctctctgatcat atggcatgga atgcagacat 2760 ctgctccgta aggcagaata tggaaggagattggaggatg acacaaaacc agcataatat 2820 cagaggaaaa gtccaaacag gacctgaactgatagaaaag ttgttactcc tggtgtagtc 2880 gcatcgacat cttgatgaac tggtggctgacacaacatac attggcttga tgtgtacata 2940 ttatttgtag ttgtgtgtgt atttttatatatatatttgt aatattgaaa tagtcataat 3000 ttactaaagg cctaccattt gccaggcatttttacatttg tcccctctaa tcttttgatg 3060 agatgatcag attggattac ttggccttgaagatgatata tctacatcta tatctatatc 3120 tatatctata tctatatcta tatctatatctatatctata tatgtatatc agaaaagctg 3180 aaatatgttt tgtaaagtta taaagatttcagactttata gaatctggga tttgccaaat 3240 gtaacccctt tctctacatt aaacccatgttggaacaaat acatttatta ttcattcatc 3300 aaatgttgct gagtcctggc tatgaaccagacactgtgaa agcctttggg atattttgcc 3360 catgcttggg caagcttata tagtttgcttcataaaactc tatttcagtt cttcataact 3420 aatacttcat gactattgct tttcaggtattccttcataa caaatacttt ggctttcata 3480 tatttgagta aagtccccct tgaggaagagtagaagaact gcactttgta aatactatcc 3540 tggaatccaa acggatagac aaggatggtgctacctcttt ctggagagta cgtgagcaag 3600 gcctgttttg ttaacatgtt ccttaggagacaaaacttag gagagacacg catagcagaa 3660 aatggacaaa aactaacaaa tgaatgggaattgtacttga ttagcattga agaccttgtt 3720 tatactatga taaatgtttg tatttgctggaagtgctact gacggtaaac cctttttgtt 3780 taaatgtgtg ccctagtagc ttgcagtatgatctattttt taagtactgt acttagctta 3840 tttaaaaatt ttatgtttaa aattgcatagtgctctttca ttgaagaagt tttgagagag 3900 agatagaatt aaattcactt atcttaccatctagagaaac ccaatgttaa aactttgttg 3960 tccattattt ctgtctttta ttcaacattttttttagagg gtgggaggaa tacagaggag 4020 gtacaatgat acacaaatga gagcactctccatgtattgt tttgtcctgt ttttcagtta 4080 acaatatatt atgagcatat ttccatttcattaaatattc ttccacaaag ttattttgat 4140 ggctgtatat caccctactt tatgaatgtaccatattaat ttatttcctg gtgtgggtta 4200 tttgatttta taatcttacc tttagaataatgaaacacct gtgaagcttt agaaaatact 4260 ggtgcctggg tctcaactcc acagattctgatttaactgg tctgggttac agactaggca 4320 ttgggaattc aaaaagttcc cccagtgattctaatgtgta gccaagatcg ggaacccttg 4380 tagacaggga tgataggagg tgagccactcttagcatcca tcatttagta ttaacatcat 4440 catcttgagt tgctaagtga atgatgcacctgacccactt tataaagaca catgtgcaaa 4500 taaaattatt ataggacttg gtttattagggcttgtgctc taagttttct atgttaagcc 4560 atacatcgca tactaaatac tttaaaatgtaccttattga catacatatt aagtgaaaag 4620 tgtttctgag ctaaacaatg acagcataattatcaagcaa tgataatttg aaatgaattt 4680 attattctgc aacttaggga caagtcatctctctgaattt tttgtacttt gagagtattt 4740 gttatatttg caagatgaag agtctgaattggtcagacaa tgtcttgtgt gcctggcata 4800 tgataggcat ttaatagttt taaagaattaatgtatttag atgaattgca taccaaatct 4860 gctgtctttt ctttatggct tcattaacttaatttgagag aaattaatta ttctgcaact 4920 tagggacaag tcatgtcttt gaatattctgtagtttgagg agaatatttg ttatatttgc 4980 aaaataaaat aagtttgcaa gttttttttttctgccccaa agagctctgt gtccttgaac 5040 ataaaataca aataaccgct atgctgttaattattggcaa atgtcccatt ttcaacctaa 5100 ggaaatacca taaagtaaca gatataccaacaaaaggtta ctagttaaca ggcattgcct 5160 gaaaagagta taaaagaatt tcagcatgattttccatatt gtgcttccac cactgccaat 5220 aaca 5224

1-58. (canceled)
 59. A method for suppressing tumor growth in a mammalcomprising: administering to said mammal the combination of areplication competent, target cell-specific adenovirus, said adenoviruscomprising an adenoviral gene essential for replication undertranscriptional control of a transcriptional regulatory element (TRE)selected from the group consisting of a probasin (PB) TRE; aprostate-specific antigen (PSA) TRE; a mucin (MUC1) TRE; anα-fetoprotein (AFP) TRE; an hKLK2 TRE; a tyrosinase TRE; a humanuroplakin II TRE (hUPII) a carcinoembryonic antigen (CEA) TRE and anE2F-1 TRE, wherein said target cell-specific adenovirus results in virusreplication-dependent cytolysis; and; at least one antineoplastic agentselected from the group consisting of 5-fluorouracil, pacitaxel,docetaxel, doxorubicin, cisplatin, estramustine and etopside, in acombined dosage effective to effect a greater reduction in the tumorcell population when compared to administration of the adenovirus vectorand antineoplastic agent alone.
 60. The method of claim 59, wherein saidat least one antineoplastic agent is selected from the group consistingof pacitaxel, docetaxel, doxorubicin, and etopside.
 61. The method ofclaim 60, wherein said at least one antineoplastic agent is pacitaxel ordocetaxel.
 62. The method of claim 59, wherein a combination ofantineoplastic agents is administered.
 63. The method of claim 59,wherein the TRE is a prostate-specific TRE selected from the groupconsisting of a PB-TRE, a PSA-TRE and an hKLK2 TRE.
 64. The method ofclaim 59, wherein said adenovirus comprises co-transcribed first andsecond genes under transcriptional control of said TRE, wherein thesecond gene is under translational control of an IRES and wherein atleast one of said first and said second genes is an adenovirus geneessential for replication.
 65. The method according to claim 59, whereinsaid adenovirus is administered by site-specific injection.
 66. Themethod according to claim 59, wherein said adenovirus is administered bysite-specific injection.
 67. The method according to claim 59, whereinsaid adenoviral gene essential for replication is an adenoviral earlygene.
 68. The method of claim 67, wherein the adenoviral early gene isE1A.
 69. The method of claim 68, wherein E1A has a mutation in ordeletion of its endogenous promoter.
 70. The method of claim 67, whereinthe adenoviral early gene is E1B.
 71. The method of claim 70, whereinE1B has a mutation in or deletion of its endogenous promoter.
 72. Themethod of claim 59, wherein E1B has a deletion of the 19-kDa region.